Rationally-designed synthetic peptide shuttle agents for delivering polypeptide cargos from an extracellular space to the cytosol and/or nucleus of a target eukaryotic cell, uses thereof, methods and kits relating to same

ABSTRACT

The present description relates to methods for delivering polypeptide cargos from an extracellular space to the cytosol and/or nucleus of a target eukaryotic cell. The methods involve contacting the cell with the polypeptide cargo in the presence of a peptide shuttle agent at a concentration sufficient to increase the polypeptide cargo&#39;s transduction efficiency. Also described here are parameters that may be used in the rational design of such synthetic peptide shuttle agents, peptide shuttle agents that satisfy one or more of these design parameters, as well as methods and compositions relating to the use of the synthetic peptide shuttle agents for delivery of a variety of polypeptide cargos (such as transcription factors, antibodies, CRISPR-associated nucleases and functional genome editing complexes) from an extracellular space to the cytosol and/or nucleus of target eukaryotic cells. Applications and targets for genome-editing NK cells for improved immunotherapy are also described.

CROSS-REFERENCE

This application is filed pursuant to 35 U.S.C. § 371 as a United StatesNational Phase Application of International Application No.PCT/CA2017/051205 filed Oct. 11, 2017, which is a Continuation-in-partof U.S. application Ser. No. 15/666,139 filed Aug. 1, 2017 which claimsbenefit of U.S. Provisional Patent Application No. 62/535,015 filed onJul. 20, 2017, and U.S. Provisional Patent Application No. 62/407,232filed on Oct. 12, 2016 which are incorporated herein by reference in itsentirety.

The present description relates to synthetic peptide shuttle agentsuseful for delivering a variety of polypeptide cargos from anextracellular space to the cytosol and/or nucleus of target eukaryoticcells. More specifically, the present description relates to parametersuseful in the rational design of such synthetic peptide shuttle agents.

BACKGROUND

Cell delivery technologies to transport large molecules insideeukaryotic cells have a wide range of applications, particularly in thebiopharmaceutical industry. While some soluble chemical substances(e.g., small molecule drugs) may passively diffuse through theeukaryotic cell membrane, larger cargos (e.g., biologics,polynucleotides, and polypeptides) require the help of shuttle agents toreach their intracellular targets.

Areas that would greatly benefit from advances in cell deliverytechnologies include the fields of genome editing and cell therapy,which have made enormous leaps over the last two decades. Decipheringthe different growth factors and molecular cues that govern cellexpansion, differentiation and reprogramming open the door to manytherapeutic possibilities for the treatment of unmet medical needs. Forexample, induction of pluripotent stem cells directly from adult cells,direct cell conversion (trans-differentiation), and genome editing (Zincfinger nuclease, TALEN and CRISPR-associated endonuclease technologies)are examples of methods that have been developed to maximize thetherapeutic value of cells for clinical applications. Presently, theproduction of cells with high therapeutic activity usually requires exvivo manipulations, mainly achieved by viral transduction, raisingimportant safety and economical concerns for human applications. Theability to directly deliver active proteins such as transcriptionfactors or artificial nucleases, inside these cells, may advantageouslycircumvent the safety concerns and regulatory hurdles associated withmore risky gene transfer methods. In particular, methods of directlydelivering active genome editing complexes in immune cells in order toimprove immunotherapy would be highly desirable.

Protein transduction approaches involving fusing a recombinant proteincargo directly to a cell-penetrating peptide (e.g., HIV transactivatingprotein TAT) require large amounts of the recombinant protein and oftenfail to deliver the cargo to the proper subcellular location, leading tomassive endosomal trapping and eventual degradation. Several endosomalmembrane-disrupting peptides have been developed to try to facilitatethe escape of endosomally-trapped cargos to the cytosol. However, manyof these endosomolytic peptides have been used to alleviate endosomalentrapment of cargos that have already been delivered intracellularly,and do not by themselves aid in the initial step of shuttling the cargosintracellularly across the plasma membrane (Salomone et al., 2012;Salomone et al., 2013; Erazo-Oliveras et al., 2014; Fasoli et al.,2014).

In particular, Salomone et al., 2012 described a chimeric peptideCM₁₈-TAT₁₁, resulting from the fusion of the Tat₁₁ cell penetratingmotif to the CM18 hybrid (residues 1-7 of Cecropin-A and 2-12 ofMelittin). This peptide was reported to be rapidly internalized by cells(due to its TAT motif) and subsequently responsible for destabilizingthe membranes of endocytic vesicles (due to the membrane disruptiveabilities of the CM18 peptide). Although the peptide CM₁₈-TAT₁₁ fused tothe fluorescent label Atto-633 (molecular weight of 774 Da; 21% of theMW of the peptide) was reported to facilitate the escape of endosomallytrapped TAT₁₁-EGFP to the cytosol (see FIG. 3 of Salomone et al., 2012),the CM₁₈-TAT₁₁ peptide (alone or conjugated to Atto-633) was not shownto act as a shuttle agent that can increase delivery of a polypeptidecargo from an extracellular space to inside of the cell—i.e., across theplasma membrane. In fact, Salomone et al., 2012 compared co-treatment(simultaneous treatment of TAT₁₁-EGFP and CM₁₈-TAT₁₁-Atto-633) versustime-shifted treatment (i.e., incubation of cells with TAT₁₁-EGFP alone,fluorescence imaging, and then incubation of the same cells with theCM₁₈-TAT₁₁-Atto-633 peptide alone, and again fluorescence imaging), andthe authors reported that “both yielded the same delivery results” (seepage 295 of Salomone et al., 2012, last sentence of first paragraphunder the heading “2.9 Cargo delivery assays”). In other words, Salomoneet al., 2012 described that the peptide CM₁₈-TAT₁₁ (alone or conjugatedto Atto-633) had no effect on delivery of a polypeptide cargo from anextracellular space to inside of the cell (i.e., protein transduction).Thus, there remains a need for improved shuttle agents capable ofincreasing the transduction efficiency of polypeptide cargos, anddelivering the cargos to the cytosol and/or nucleus of target eukaryoticcells.

The present description refers to a number of documents, the content ofwhich is herein incorporated by reference in their entirety.

SUMMARY

A plurality of different peptides was screened with the goal ofidentifying polypeptide-based shuttle agents that can deliverindependent polypeptide cargos intracellularly to the cytosol/nucleus ofeukaryotic cells. On one hand, these large-scale screening efforts ledto the surprising discovery that certain domain-based peptide shuttleagents increase the transduction efficiency of polypeptide cargos ineukaryotic cells, by increasing the number and/or proportion of cellsthat ultimately internalize the polypeptide cargos, and also enable theinternalized cargos to gain access to the cytosol/nuclear compartment(thus avoiding or reducing cargo endosomal entrapment). Thesedomain-base shuttle agents comprise an endosome leakage domain (ELD)operably linked to a cell penetrating domain (CPD), and optionally oneor more histidine-rich domains. On the other hand, the above screeningefforts also revealed some peptides having no or low polypeptide cargotransduction activity, excessive toxicity, and/or other undesirableproperties (e.g., poor solubility and/or stability). These empiricaldata (both positive and negative) were used herein to identifyphysiochemical properties of successful, less successful, and failedpeptides in order to arrive at a set of design parameters that enablethe rational design and/or identification of peptides having proteintransduction activity.

Accordingly, the present description relates to methods for deliveringpolypeptide cargos from an extracellular space to the cytosol and/ornucleus of a target eukaryotic cell by contacting the cell with thepolypeptide cargo in the presence of a peptide shuttle agent asdescribed herein, at a concentration sufficient to increase thepolypeptide cargo's transduction efficiency, as compared to in theabsence of the shuttle agent. More particularly, the present descriptionrelates to parameters that may be used in the rational design of suchsynthetic peptide shuttle agents, peptide shuttle agents that satisfyone or more of these design parameters, as well as methods andcompositions relating to the use of the synthetic peptide shuttle agentsfor delivery of a variety of polypeptide cargos from an extracellularspace to the cytosol and/or nucleus of target eukaryotic cells. Thepresent description also relates to machine-learning orcomputer-assisted approaches that may be used to generate peptidevariants that respect one or more of the design parameters describedherein.

The present description also relates to co-transducing a polypeptidecargo of interest and a marker protein as a means to identify and/orenrich transduced cells. It was surprisingly discovered that astrikingly high proportion of target eukaryotic cells that weresuccessfully transduced with a polypeptide cargo of interest, were alsosuccessfully transduced with a marker protein. Conversely, a strikinglyhigh proportion of cells that were not transduced with the polypeptidecargo of interest, were also not transduced with the marker protein.Isolating cells positive for the marker protein (e.g., via FACS)resulted in a significant increase in the proportion of cells that weresuccessfully transduced with the polypeptide cargo of interest, and thecorrelation was found to be concentration dependent in that cellpopulations exhibiting the highest fluorescence of the marker proteinalso tended to exhibit the highest proportion of transduction with thepolypeptide cargo of interest. It was also discovered that cells thatwere unsuccessfully transduced following a first round of transductionwith a polypeptide cargo of interest, may be isolated and re-transducedwith the polypeptide cargo of interest in subsequent rounds oftransduction. Thus, in some aspects, the present description relates tomethods comprising co-transduction of a polypeptide cargo of interestwith a marker protein, wherein the marker protein may be used to isolateor enrich cells transduced with a polypeptide cargo of interest. In someembodiments, the present description also relates to methods comprisingrepeated successive transduction experiments performed on, for example,cells that were not successfully transduced with a marker proteinfollowing a first or previous transduction reaction. Such methodspresent attractive approaches for increasing transduction efficiency invaluable cell populations (e.g., patient-derived cells for celltherapy), and/or in cell populations that are inherently more difficultto transduce.

General Definitions

Headings, and other identifiers, e.g., (a), (b), (i), (ii), etc., arepresented merely for ease of reading the specification and claims. Theuse of headings or other identifiers in the specification or claims doesnot necessarily require the steps or elements be performed inalphabetical or numerical order or the order in which they arepresented.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one” butit is also consistent with the meaning of “one or more”, “at least one”,and “one or more than one”.

The term “about” is used to indicate that a value includes the standarddeviation of error for the device or method being employed in order todetermine the value. In general, the terminology “about” is meant todesignate a possible variation of up to 10%. Therefore, a variation of1, 2, 3, 4, 5, 6, 7, 8, 9 and 10% of a value is included in the term“about”. Unless indicated otherwise, use of the term “about” before arange applies to both ends of the range.

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

As used herein, “protein” or “polypeptide” or “peptide” means anypeptide-linked chain of amino acids, which may or may not comprise anytype of modification (e.g., chemical or post-translational modificationssuch as acetylation, phosphorylation, glycosylation, sulfatation,sumoylation, prenylation, ubiquitination, etc.). For further clarity,protein/polypeptide/peptide modifications are envisaged so long as themodification does not destroy the protein transduction activity of theshuttle agents described herein

As used herein, a “domain” or “protein domain” generally refers to apart of a protein having a particular functionality or function. Somedomains conserve their function when separated from the rest of theprotein, and thus can be used in a modular fashion. The modularcharacteristic of many protein domains can provide flexibility in termsof their placement within the shuttle agents of the present description.However, some domains may perform better when engineered at certainpositions of the shuttle agent (e.g., at the N- or C-terminal region, ortherebetween). The position of the domain within its endogenous proteinis sometimes an indicator of where the domain should be engineeredwithin the shuttle agent and of what type/length of linker should beused. Standard recombinant DNA techniques can be used by the skilledperson to manipulate the placement and/or number of the domains withinthe shuttle agents of the present description in view of the presentdisclosure. Furthermore, assays disclosed herein, as well as othersknown in the art, can be used to assess the functionality of each of thedomains within the context of the shuttle agents (e.g., their ability tofacilitate cell penetration across the plasma membrane, endosome escape,and/or access to the cytosol). Standard methods can also be used toassess whether the domains of the shuttle agent affect the activity ofthe cargo to be delivered intracellularly. In this regard, theexpression “operably linked” as used herein refers to the ability of thedomains to carry out their intended function(s) (e.g., cell penetration,endosome escape, and/or subcellular targeting) within the context of theshuttle agents of the present description. For greater clarity, theexpression “operably linked” is meant to define a functional connectionbetween two or more domains without being limited to a particular orderor distance between same.

As used herein, the term “synthetic” used in expressions such as“synthetic peptide” or “synthetic polypeptide” is intended to refer tonon-naturally occurring molecules that can be produced in vitro (e.g.,synthesized chemically and/or produced using recombinant DNAtechnology). The purities of various synthetic preparations may beassessed by, for example, high-performance liquid chromatographyanalysis and mass spectroscopy. Chemical synthesis approaches may beadvantageous over cellular expression systems (e.g., yeast or bacteriaprotein expression systems), as they may preclude the need for extensiverecombinant protein purification steps (e.g., required for clinicaluse). In contrast, longer synthetic polypeptides may be more complicatedand/or costly to produce via chemical synthesis approaches and suchpolypeptides may be more advantageously produced using cellularexpression systems. In some embodiments, the peptides or shuttle agentof the present description may be chemically synthesized (e.g., solid-or liquid phase peptide synthesis), as opposed to expressed from arecombinant host cell. In some embodiments, the peptides or shuttleagent of the present description may lack an N-terminal methionineresidue. A person of skill in the art may adapt a synthetic peptide orshuttle agent of the present description by using one or more modifiedamino acids (e.g., non-naturally-occurring amino acids), or bychemically modifying the synthetic peptide or shuttle agent of thepresent description, to suit particular needs of stability or otherneeds.

The expression “polypeptide-based” when used here in the context of ashuttle agent of the present description, is intended to distinguish thepresently described shuttle agents from non-polypeptide ornon-protein-based shuttle agents such as lipid- or cationicpolymer-based transduction agents, which are often associated withincreased cellular toxicity and may not be suitable for use in humantherapy.

As used herein, the term “independent” is generally intended refer tomolecules or agents which are not covalently bound to one another. Forexample, the expression “independent polypeptide cargo” is intended torefer to a polypeptide cargo to be delivered intracellularly that is notcovalently bound (e.g., not fused) to a shuttle agent of the presentdescription. In some aspects, having shuttle agents that are independentof (not fused to) a polypeptide cargo may be advantageous by providingincreased shuttle agent versatility—e.g., not being required tore-engineer a new fusion protein for different polypeptide cargoes,and/or being able to readily vary the ratio of shuttle agent to cargo(as opposed to being limited to a 1:1 ratio in the case of a fusionprotein).

As used herein, the expression “is or is from” or “is from” comprisesfunctional variants of a given protein domain (e.g., CPD or ELD), suchas conservative amino acid substitutions, deletions, modifications, aswell as variants or function derivatives, which do not abrogate theactivity of the protein domain.

Other objects, advantages and features of the present description willbecome more apparent upon reading of the following non-restrictivedescription of specific embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

In the appended drawings:

FIGS. 1A and 1B show a typical result of a calcein endosomal escapeassay in which HEK293A cells were loaded with the fluorescent dyecalcein (“100 μM calcein”), and were then treated (or not) with ashuttle agent that facilitates endosomal escape of the calcein (“100 μMcalcein+CM18-TAT 5 μM”). FIG. 1A shows the results of a fluorescencemicroscopy experiment, while FIG. 1B shows the results of a flowcytometry experiment.

FIG. 2 shows the results of a calocein endosomal escape flow cytometryassay in which HeLa cells were loaded with calcein (“calcein 100 μM”),and were then treated with increasing concentrations of the shuttleagent CM18-TAT-Cys (labeled “CM18-TAT”).

FIGS. 3 and 4 show the results of calcein endosomal escape flowcytometry assays in which HeLa cells (FIG. 3) or primary myoblasts (FIG.4) were loaded with calcein (“calcein 100 μM”), and were then treatedwith 5 μM or 8 μM of the shuttle agents CM18-TAT-Cys orCM18-Penetratin-Cys (labeled “CM18-TAT” and “CM18-Penetratin”,respectively).

FIG. 5 shows the results of a GFP transduction experiment visualized byfluorescence microscopy in which a GFP cargo protein was co-incubatedwith 0, 3 or 5 μM of CM18-TAT-Cys (labeled “CM18-TAT”), and then exposedto HeLa cells. The cells were observed by bright field (upper panels)and fluorescence microscopy (lower panels).

FIGS. 6A and 6B show the results of a GFP transduction efficiencyexperiment in which GFP cargo protein (10 μM) was co-incubated withdifferent concentrations of CM18-TAT-Cys (labeled “CM18-TAT”), prior tobeing exposed to HeLa cells. Cells were evaluated by flow cytometry andthe percentage of fluorescent (GFP-positive) cell is shown in FIG. 6A,and corresponding cell toxicity data is shown in FIG. 6B.

FIGS. 7A and 7B shows the results of a GFP transduction efficiencyexperiment in which different concentrations of GFP cargo protein (10, 5or 1 μM) were co-incubated with either 5 μM of CM18-TAT-Cys (FIG. 7A,labeled “CM18TAT”), or 2.5 μM of dCM18-TAT-Cys (FIG. 7B, labeled“dCM18TAT”), prior to being exposed to HeLa cells. Cells were evaluatedby flow cytometry and the percentages of fluorescent (GFP-positive)cells are shown.

FIGS. 8 and 9 show the results of GFP transduction efficiencyexperiments in which GFP cargo protein (10 μM) was co-incubated withdifferent concentrations and combinations of CM18-TAT-Cys (labeled“CM18TAT”), CM18-Penetratin-Cys (labeled “CM18penetratin”), and dimersof each (dCM18-TAT-Cys (labeled “dCM18TAT”), dCM18-Penetratin-Cys(labeled “dCM18penetratin”), prior to being exposed to HeLa cells. Cellswere evaluated by flow cytometry and the percentages of fluorescent(GFP-positive) cells are shown.

FIG. 10 shows typical results of a TAT-GFP transduction experiment inwhich TAT-GFP cargo protein (5 μM) was co-incubated with 3 μM ofCM18-TAT-Cys (labeled “CM18-TAT”), prior to being exposed to HeLa cells.Cells and GFP fluorescence were visualized by bright field andfluorescence microscopy at 10× and 40× magnifications. Arrows indicatethe endosome delivery of TAT-GFP in the absence of CM18-TAT-Cys, as wellas its nuclear delivery in the presence of CM18-TAT-Cys.

FIGS. 11A and 11B show the results of a TAT-GFP transduction efficiencyexperiment in which TAT-GFP cargo protein (5 μM) was co-incubated withdifferent concentrations of CM18-TAT-Cys (labeled “CM18TAT”), prior tobeing exposed to HeLa cells. Cells were evaluated by flow cytometry andthe percentage of fluorescent (GFP-positive) cell is shown in FIG. 11A,and corresponding cell toxicity data is shown in FIG. 11B.

FIG. 12 shows typical results of a GFP-NLS transduction experiment inwhich GFP-NLS cargo protein (5 μM) was co-incubated with 5 μM ofCM18-TAT-Cys (labeled “CM18-TAT”), prior to being exposed to HeLa cellsfor 5 minutes. Cells and GFP fluorescence were visualized by brightfield and fluorescence microscopy at 10×, 20×, and 40× magnifications.Arrows indicate areas of nuclear delivery of GFP-NLS.

FIGS. 13A and 13B show the results of a GFP-NLS transduction efficiencyexperiment in which GFP-NLS cargo protein (5 μM) was co-incubated withdifferent concentrations of CM18-TAT-Cys (labeled “CM18TAT”), prior tobeing exposed to HeLa cells. Cells were evaluated by flow cytometry andthe percentage of fluorescent (GFP-positive) cell is shown in FIG. 13A,and corresponding cell toxicity data is shown in FIG. 13B.

FIGS. 14 and 15 show the results of GFP-NLS transduction efficiencyexperiments in which GFP-NLS cargo protein (5 μM) was co-incubated withdifferent concentrations and combinations of CM18-TAT (labeled“CM18TAT”), CM18-Penetratin (labeled “CM18penetratin”), and dimers ofeach (dCM18-TAT-Cys, dCM18-Penetratin-Cys; labeled “dCM18TAT” and“dCM18penetratin”, respectively), prior to being exposed to HeLa cells.Cells were evaluated by flow cytometry and the percentages offluorescent (GFP-positive) cells are shown.

FIG. 16 shows the results of a GFP-NLS transduction efficiencyexperiment in which GFP-NLS cargo protein (5 μM) was co-incubated witheither CM18-TAT-Cys (3.5 μM, labeled “CM18TAT”) alone or withdCM18-Penetratin-Cys (1 μM, labeled “dCM18pen”) for 5 minutes or 1 hourin plain DMEM media (“DMEM”) or DMEM media containing 10% FBS (“FBS”),before being subjected to flow cytometry analysis. The percentages offluorescent (GFP-positive) cells are shown. Cells that were not treatedwith shuttle agent or GFP-NLS (“ctrl”), and cells that were treated withGFP-NLS without shuttle agent (“GFP-NLS 5 μM”) were used as controls.

FIGS. 17A and 17B show the results of a GFP-NLS transduction efficiencyexperiment in which GFP-NLS cargo protein (5 μM) was co-incubated withor without 1 μM CM18-TAT-Cys (labeled “CM18TAT”), prior to being exposedto THP-1 cells. Cells were evaluated by flow cytometry and thepercentage of fluorescent (GFP-positive) cells is shown in FIG. 17A, andcorresponding cell toxicity data is shown in FIG. 17B.

FIG. 18 shows the results of a transduction efficiency experiment inwhich the cargo protein, FITC-labeled anti-tubulin antibody (0.5 μM),was co-incubated with 5 μM of CM18-TAT-Cys (labeled “CM18-TAT”), priorto being exposed to HeLa cells. Functional antibody delivery wasvisualized by bright field (20×) and fluorescence microscopy (20× and40×), in which fluorescent tubulin fibers in the cytoplasm werevisualized.

FIGS. 19A and 19B shows the results of an FITC-labeled anti-tubulinantibody transduction efficiency experiment in which the antibody cargoprotein (0.5 μM) was co-incubated with 3.5 μM of CM18-TAT-Cys (labeled“CM18TAT”), CM18-Penetratin-Cys (labeled “CM18pen”) ordCM18-Penetratin-Cys (labeled “dCM18pen”), or a combination of 3.5 μM ofCM18-TAT-Cys and 0.5 μM of dCM18-Penetratin-Cys, prior to being exposedto HeLa cells. Cells were evaluated by flow cytometry and the percentageof fluorescent (FITC-positive) cell is shown in FIG. 19A, andcorresponding cell toxicity data is shown in FIG. 19B.

FIG. 20 shows the results of DNA transfection efficiency experiment inwhich plasmid DNA (pEGFP) was labeled with a Cy5™ dye was co-incubatedwith 0, 0.05, 0.5, or 5 μM of CM18-TAT-Cys (labeled “CM18-TAT”), priorto being exposed to HEK293A cells. Flow cytometry analysis allowedquantification of Cy5™ emission (corresponding to DNA intracellulardelivery; y-axis) and GFP emission (corresponding to successful nucleardelivery of DNA; percentage indicated above each bar).

FIGS. 21A and 21B show the results of a GFP-NLS transduction efficiencyexperiment in which the GFP-NLS cargo protein (5 μM) was co-incubatedwith 1, 3, or 5 μM of CM18-TAT-Cys (labeled “CM18TAT”), of His-CM18-TAT(labeled “His-CM18TAT”), prior to being exposed to HeLa cells. Cellswere evaluated by flow cytometry and the percentage of fluorescent(GFP-positive) cell is shown in FIG. 21A, and corresponding celltoxicity data is shown in FIG. 21B.

FIGS. 22A and 22B show the results of a transduction efficiencyexperiment in which GFP-NLS cargo protein was intracellularly deliveredusing the shuttle His-CM18-PTD4 in HeLa cells. GFP-NLS transductionefficiency was evaluated by flow cytometry and the percentage of GFPfluorescent cells (“Pos cells (%)”), as well as corresponding cellviability data (“viability (%)”) are shown. FIG. 22A shows a comparisonof GFP-NLS transduction efficiencies using different transductionprotocols (Protocol A vs. B). FIG. 22B shows the effect of usingdifferent concentrations of the shuttle His-CM18-PTD4 when usingProtocol B.

FIGS. 23-26 are microscopy images showing the results of transductionexperiments in which GFP-NLS (FIGS. 23, 24A, 24B, 25 and 26) orFITC-labeled anti-tubulin antibody (FIGS. 24C and 24D) cargo protein wasintracellularly delivered with the shuttle His-CM18-PTD4 in HeLa cells.The bright field and fluorescence images of living cells are shown inFIGS. 23, 24 and 26. In FIG. 25, the cells were fixed, permeabilized andsubjected to immuno-labelling with an anti-GFP antibody and afluorescent secondary antibody. White triangle windows indicate examplesof areas of co-labelling between nuclei (DAPI) and GFP-NLS signals. FIG.26 shows images captured by confocal microscopy.

FIGS. 27A-27D show microscopy images of a kinetic (time-course)transduction experiment in HeLa cells, where the fluorescence of GFP-NLScargo protein was tracked after 45 (FIG. 27A), 75 (FIG. 27B), 100 (FIG.27C), and 120 (FIG. 27D) seconds following intracellular delivery withthe shuttle His-CM18-PTD4. The diffuse cytoplasmic fluorescence patternobserved after 45 seconds (FIG. 27A) gradually becomes a moreconcentrated nuclear pattern at 120 seconds (FIG. 27D).

FIGS. 28A-28D show microscopy images of co-delivery transductionexperiment in which two cargo proteins (GFP-NLS and mCherry™-NLS) aresimultaneously delivered intracellularly by the shuttle His-CM18-PTD4 inHeLa cells. Cells and fluorescent signals were visualized by (FIG. 28A)bright field and (FIG. 28B-28D) fluorescence microscopy. White trianglewindows indicate examples of areas of co-labelling between nuclei (DAPI)and GFP-NLS or mCherry™.

FIGS. 29A-29I show the results of GFP-NLS transduction efficiencyexperiments in HeLa cells using different shuttle agents orsingle-domain/control peptides. GFP-NLS transduction efficiency wasevaluated by flow cytometry and the percentage of GFP fluorescent cells(“Pos cells (%)”), as well as corresponding cell viability data(“viability (%)”) are shown in FIGS. 29A, 29B, 29D-29G and 29I. In FIGS.29A and 29D-29F, cells were exposed to the cargo/shuttle agent for 10seconds. In panel I, cells were exposed to the cargo/shuttle agent for 1minute. In FIGS. 29B, 29C, 29G and 29H, cells were exposed to thecargo/shuttle agent for 1, 2, or 5 min. “Relative fluorescence intensity(FL1-A)” or “Signal intensity” corresponds to the mean of allfluorescence intensities from each cell with a GFP fluorescent signalafter GFP-NLS fluorescent protein delivery with the shuttle agent. FIG.29D shows the results of a control experiment in which onlysingle-domain peptides (ELD or CDP) or the peptide His-PTD4 (His-CPD)were used for the GFP-NLS transduction, instead of the multi-domainshuttle agents.

FIGS. 30A-30F show microscopy images of HeLa cells transduced withGFP-NLS using the shuttle agent FIG. 30A: TAT-KALA, FIG. 30B:His-CM18-PTD4, FIG. 30C: His-C(LLKK)₃C-PTD4, FIG. 300: PTD4-KALA, FIG.30E: EB1-PTD4, and FIG. 30F: His-CM18-PTD4-His. The insets in the bottomrow panels show the results of corresponding flow cytometry analyses,indicating the percentage of cells exhibiting GFP fluorescence.

FIG. 31 shows the results of a transduction efficiency experiment inwhich GFP-NLS cargo protein was intracellularly delivered using theshuttle His-CM18-PTD4 in THP-1 cells using different Protocols (ProtocolA vs C). GFP-NLS transduction efficiency was evaluated by flow cytometryand the percentage of GFP fluorescent cells (“Pos cells (%)”), as wellas corresponding cell viability data (“viability (%)”) are shown. “Ctrl”corresponds to THP-1 cells exposed to GFP-NLS cargo protein in theabsence of a shuttle agent.

FIGS. 32A-32D shows microscopy images of THP-1 cells transduced withGFP-NLS cargo protein using the shuttle His-CM18-PTD4. Images capturedunder at 4×, 10× and 40× magnifications are shown in FIGS. 32A-32C,respectively. White triangle windows in FIG. 32C indicate examples ofareas of co-labelling between cells (bright field) and GFP-NLSfluorescence. FIG. 32D shows the results of corresponding flow cytometryanalyses, indicating the percentage of cells exhibiting GFPfluorescence.

FIG. 33A-33D show microscopy images of THP-1 cells transduced withGFP-NLS cargo protein using the shuttle His-CM18-PTD4. White trianglewindows indicate examples of areas of co-labelling between cells (brightfield; FIGS. 33A and 33B), and GFP-NLS fluorescence (FIG. 33C and FIG.33D).

FIGS. 34A-34B show the results of GFP-NLS transduction efficiencyexperiments in THP-1 cells using the shuttle TAT-KALA, His-CM18-PTD4, orHis-C(LLKK)₃C-PTD4. The cargo protein/shuttle agents were exposed to theTHP-1 cells for 15, 30, 60 or 120 seconds. GFP-NLS transductionefficiency was evaluated by flow cytometry and the percentage of GFPfluorescent cells (“Pos cells (%)”), as well as corresponding cellviability data (“viability (%)”) are shown in FIG. 34A. In FIG. 348,“Relative fluorescence intensity (FL1-A)” corresponds to the mean of allfluorescence intensities from each cell with a GFP fluorescent signalafter GFP-NLS fluorescent protein delivery with the shuttle agent.

FIG. 35A-35F shows the results of transduction efficiency experiments inwhich THP-1 cells were exposed daily to GFP-NLS cargo in the presence ofa shuttle agent for 2.5 hours. His-CM18-PTD4 was used in FIGS. 35A-35E,and His-C(LLKK)₃C-PTD4 was used in FIG. 35F. GFP-NLS transductionefficiency was determined by flow cytometry at Day 1 or Day 3, and theresults are expressed as the percentage of GFP fluorescent cells (“Poscells (%)”), as well as corresponding cell viability data (“viability(%)”) in FIGS. 35A-35C and 35F. FIG. 35D shows the metabolic activityindex of the THP-1 cells after 1, 2, 4, and 24 h, and FIG. 35E shows themetabolic activity index of the THP-1 cells after 1 to 4 days, for cellsexposed to the His-CM18-PTD4 shuttle.

FIG. 36 shows a comparison of the GFP-NLS transduction efficiencies in aplurality of different types of cells (e.g., adherent and suspension, aswell as cell lines and primary cells) using the shuttle His-CM18-PTD4,as measured by flow cytometry. The results are expressed as thepercentage of GFP fluorescent cells (“Pos cells (%)”), as well ascorresponding cell viability data (“viability (%)”).

FIGS. 37A-37H show fluorescence microscopy images of different types ofcells transduced with GFP-NLS cargo using the shuttle His-CM18-PTD4. GFPfluorescence was visualized by fluorescence microscopy at a 10×magnification. The results of parallel flow cytometry experiments arealso provided in the insets (viability and percentage of GFP-fluorescingcells).

FIGS. 38A-38B show fluorescence microscopy images of primary humanmyoblasts transduced with GFP-NLS using the shuttle His-CM18-PTD4. Cellswere fixed and permeabilized prior to immuno-labelling GFP-NLS with ananti-GFP antibody and a fluorescent secondary antibody. Immuno-labelledGFP is shown in FIG. 38A, and this image is overlaid with nuclei (DAPI)labelling in FIG. 38B.

FIG. 39A-39E show a schematic layout (FIGS. 39A, 39B and 39C) and samplefluorescence images (FIGS. 39D and 39E) of a transfection plasmidsurrogate assay used to evaluate the activity of intracellularlydelivered CRISPR/Cas9-NLS complex. At Day 1 (FIG. 39A), cells aretransfected with an expression plasmid encoding the fluorescent proteinsmCherry™ and GFP, with a STOP codon separating their two open readingframes. Transfection of the cells with the expression plasmid results inonly mCherry™ expression (FIG. 39D). A CRISPR/Cas9-NLS complex, whichhas been designed/programmed to cleave the plasmid DNA at the STOPcodon, is then delivered intracellularly to the transfected cellsexpressing mCherry™, resulting double-stranded cleavage of the plasmidDNA at the STOP codon (FIG. 39B). In a fraction of the cells, randomnon-homologous DNA repair of the cleaved plasmid occurs and results inremoval of the STOP codon (FIG. 39C), and thus GFP expression andfluorescence (FIG. 39E). White triangle windows indicate examples ofareas of co-labelling of mCherry™ and GFP fluorescence.

FIG. 40A-40H show fluorescence microscopy images of HeLa cellsexpressing mCherry™ and GFP, indicating CRISPR/Cas9-NLS-mediatedcleavage of plasmid surrogate DNA. In panels A-D, HeLa cells wereco-transfected with three plasmids: the plasmid surrogate as describedin the brief description of FIGS. 39A-39E, and two other expressionplasmids encoding the Cas9-NLS protein and crRNA/tracrRNAs,respectively. CRISPR/Cas9-mediated cleavage of the plasmid surrogate atthe STOP codon, and subsequent DNA repair by the cell, enablesexpression of GFP (FIGS. 40B and 40D) in addition to mCherry™ (FIGS. 40Aand 40C). In FIGS. 40E-40H, HeLa cells were transfected with the plasmidsurrogate and then transduced with an active CRISPR/Cas9-NLS complexusing the shuttle His-CM18-PTD4. CRISPR/Cas9-NLS-mediated cleavage ofthe plasmid surrogate at the STOP codon, and subsequent DNA repair bythe cell, enables expression of GFP (FIGS. 40F and 40H) in addition tomCherry™ (panels 40E and 40G).

FIG. 41A (lanes A to D) shows the products of a DNA cleavage assay (T7E1assay) separated by agarose gel electrophoresis, which is used tomeasure CRISPR/Cas9-mediated cleavage of cellular genomic DNA. HeLacells were transduced with a CRISPR-Cas9-NLS complex programmed tocleave the PPIB gene. The presence of the cleavage product framed inwhite boxes 1 and 2, indicates cleavage of the PPIB gene by theCRISPR-Cas9-NLS complex, which was delivered intracellularly using theshuttle His-C(LLKK)₃C-PTD4 (FIG. 41A, lane B) or with a lipidictransfection agent used as a positive control (FIG. 41A, lane D). Thiscleavage product is absent in negative controls (FIG. 41A, lanes A andC).

FIG. 41B shows the products of a DNA cleavage assay (T7E1 assay)separated by agarose gel electrophoresis, which is used to measureCRISPR/Cas9-mediated cleavage of cellular genomic DNA (PPIB DNAsequences). The left panel shows the cleavage product of the amplifiedPPIB DNA sequence by the CRIPR/Cas9 complex after the delivery of thecomplex with the shuttle agent His-CM18-PTD4 in HeLa cells. The rightpanel shows amplified DNA sequence before the T7E1 digestion procedureas a negative control.

FIG. 41C shows the products of a DNA cleavage assay (T7E1 assay)separated by agarose gel electrophoresis, which is used to measureCRISPR/Cas9-mediated cleavage of cellular genomic DNA (PPIB DNAsequences). The left panel shows the amplified PPIB DNA sequence afterincubation of the HeLa cells with the Cas9/RNAs complex in presence of alipidic transfection agent (DharmaFect™ transfection reagent #T-20XX-01) (positive control). The right panel shows amplified DNAsequence before the T7E1 digestion procedure as a negative control.FIGS. 42-44 show the transcriptional activity of THP-1 cells that havebeen transduced with the transcription factor HOXB4 using differentconcentrations of the shuttle His-CM18-PTD4 and different cargo/shuttleexposure times. Successful intra-nuclear delivery of HOXB4 wasdetermined by monitoring mRNA levels of a target gene by real-time PCR,and the results are normalized against those in the negative control(HOXB4 without shuttle agent) and expressed as “Fold over control” (leftbars). Total cellular RNA (ng/μL) was quantified and used a marker forcell viability (right bars). “Ø” or “Ctrl” means “no treatment”; “TF”means “Transcription Factor alone”; “FS” means “shuttle alone”.

FIGS. 45A-45D show fluorescence microscopy images of HeLa cellstransduced with wild-type HOXB4 cargo using the shuttle His-CM18-PTD4.After a 30-minute incubation to allow transduced HOXB4-WT to accumulatein the nucleus, the cells were fixed, permeabilized and HOXB4-WT waslabelled using a primary anti-HOXB4 monoclonal antibody and afluorescent secondary antibody (FIGS. 45B and 451). Nuclei were labelledwith DAPI (FIGS. 45A and 45C). White triangle windows indicate examplesof areas of co-labelling between nuclei and HOXB4—compare FIG. 45A vs45B (×20 magnification), and FIG. 45C vs 45D (×40 magnification).

FIGS. 46A and 46B show the products of a DNA cleavage assay separated byagarose gel electrophoresis, which is used to measureCRISPR/Cas9-mediated cleavage of cellular genomic DNA (HPTR sequence)after intracellular delivery of the complex with different shuttleagents. FIG. 46A shows the results with the shuttle agents:His-CM18-PTD4, His-CM18-PTD4-His, and His-C(LLKK)3C-PTD4 in HeLa cells.FIG. 46B shows the results with His-CM18-PTD4-His and His-CM18-L2-PTD4in Jurkat cells. Negative controls (lane 4 in panels A and B) showamplified HPTR DNA sequence after incubation of the cells with theCRISPR/Cas9 complex without the presence of the shuttle agent. Positivecontrols (lane 5 in panels A and B) show the amplified HPTR DNA sequenceafter incubation of the cells with the Cas9/RNAs complex in presence ofa commercial lipidic transfection agent.

FIG. 47 shows the transcriptional activity of THP-1 cells that have beentransduced with the transcription factor HOXB4 using the shuttle agentsHis-CM18-PTD4, TAT-KALA, EB1-PTD4, His-C(LLKK)3C-PTD4 andHis-CM18-PTD4-His. Successful intra-nuclear delivery of HOXB4 wasdetermined by monitoring mRNA levels of a target gene by real-time PCR,and the results were normalized against those in the negative control(HOXB4 without shuttle agent) and expressed as “Fold over control” (leftbars). Total cellular RNA (ng/μL) was quantified and used a marker forcell viability (right bars). “Ø” or “Ctrl” means “no treatment”; “TF”means “Transcription Factor alone”; “FS” means “shuttle alone”.

FIGS. 48A-48D show in vivo GFP-NLS delivery in rat parietal cortex byHis-CM18-PTD4. Briefly, GFP-NLS (20 μM) was injected in the parietalcortex of rat in presence of the shuttle agent His-CM18-PTD4 (20 μM) for10 min. Dorso-ventral rat brain slices were collected and analyzed byfluorescence microscopy at 4× (FIG. 48A), 10× (FIG. 48C) and 20×magnifications (FIG. 48D). The injection site is located in the deepestlayers of the parietal cortex (PCx). In presence of the His-CM18-PTD4shuttle agent, the GFP-NLS diffused in cell nuclei of the PCx, of theCorpus Callus (Cc) and of the striatum (Str) (white curves marklimitations between brains structures). FIG. 48B shows the stereotaxiccoordinates of the injection site (black arrows) from the rat brainatlas of Franklin and Paxinos. The injection of GFP-NLS in presence ofHis-CM18-PTD4 was performed on the left part of the brain, and thenegative control (injection of GFP-NLS alone), was done on thecontralateral site. The black circle and connected black lines in FIG.48B show the areas observed in the fluorescent pictures (FIGS. 48A, 48Cand 48D)

FIGS. 49A and 49B show helical wheel (left panels) and “open cylinder”(right panels) representations of the peptides FSD5 and VSVG-PTD4,respectively. The geometrical shape of each amino acid residuecorresponds to its biochemical property based on the residue's sidechain (i.e., hydrophobicity, charge, or hydrophilicity). One of the maindifferences between the two opened cylindrical representations of FSD5and VSVG-PTD4 is the presence of a highly hydrophobic core in FSD5(outlined in FIG. 49A, left and right panels), which is not present inVSVG-PTD4. The cylinder in the lower middle panels of FIGS. 49A and 49Brepresent simplified versions of the opened cylindrical representationsin the right panels, in which: “H” represents the high hydrophobicsurface area; “h” represents low hydrophobic surface area; “+”represents positively charged residues; and “h” represent hydrophilicresidues.

FIGS. 49C-49F show predicted 3-dimensional models of the structures ofthe peptides FSD5, FSD18, VSVG-PTD4, and a peptide having twobeta-sheets and no alpha helices, respectively.

FIG. 49G shows a multiple sequence alignment of the peptidesHis-CM18-PTD4; EB1-PTD4; His-C(LLKK)₃C-PTD4; FSD5; FSD10; FSD19; FSD20;FSD21; FSD44; FSD46; and FSD63, along with “Consistency” scores for eachresidue position. For the alignment, histidine-rich domains werevoluntarily excluded.

FIGS. 50A-50C show microscopy images of live HeLa cells successfullytransduced by the shuttle agent FSD5 with fluorescently labelledantibodies as cargos. FIG. 50A shows the cytoplasmic transduction of aGoat Anti-Rabbit IgG H&L (Alexa Fluor 594) antibody visualized by brightfield (upper panel) and fluorescence microscopy (lower panel) at 20×magnification. FIGS. 50B and 50C show the cytoplasmic transduction of aGoat Anti-Mouse IgG H&L (Alexa Fluor® 488) antibody visualized by brightfield (upper panels) and fluorescence microscopy (lower panels) at 10×and 20× magnifications, respectively.

FIG. 50D shows the results of a transduction experiment in which ananti-NUP98 antibody, which recognizes an antigen in the perinuclearmembrane, was transduced into HeLa cells using the shuttle agent FSD19.Following transduction, HeLa cells were fixed, permeabilized andlabelled with a fluorescent (Alexa™ 488) secondary antibody recognizingthe anti-NUP98 antibody (left panels) and Hoechst nuclear staining(right panels). Upper and lower panels indicate images taken under 20×and 40× magnification, respectively.

FIGS. 51A-51F show the results of genome editing experiments in whichCRISPR/Cas9-NLS genome editing complexes were transduced into differentcell types (HeLa, NK cells, NIC-H196 cells, HCC-78 cells, and REC-1cells) using different shuttle agent peptides (FSD5, FSD8, FSD10,FSD18), and successful genome editing was verified by genomic DNAcleavage assays. The CRISPR/Cas9-NLS complexes consisted of recombinantCas9-NLS complexed with a crRNA/tracrRNA designed to cleave the HPRTgenomic DNA sequence. Successful genome editing was observed by thedetection of genomic DNA cleavage products (thick solid arrows), ascompared to the uncleaved genomic target gene (thin dashed arrows). Thenegative control (− ctrl”) were from transduction experiments performedin the absence of any shuttle agent peptide. An imaging software wasused to quantify the relative signal intensities of the cleavage productbands directly on gels. The sum of all the bands in a given lanecorresponds to 100% of the signal, and the numerical value in italics atthe bottom of each lane is the sum of the relative signals (%) of onlythe two cleavage product bands (thick solid arrows).

FIG. 51G shows the cleavage of the targeted HPRT genomic sequence by theCRISPR/Cas9-NLS complex transduced by FSD5, in the absence (“Notemplate”) or presence (“+500 ng”) of a short DNA template (72 bp). Thindashed arrows indicate the bands corresponding to the target gene, andthick solid arrows indicate the bands corresponding to theCRISPR/Cas9-NLS-mediated cleavage products of this target gene, whichindicate the successful transduction of fully functional genome editingcomplexes in the presence and absence of the DNA template. The numericalvalue in italics at the bottom of each lane is the sum of the relativesignals (%) of only the two cleavage product bands (thick solid arrows).FIG. 51H shows the results of a PCR-amplification of the samples of FIG.51G, using primers specific for the short DNA template, indicatinggenomic insertion of the DNA template sequence (see arrow in FIG. 51H).

FIG. 51I shows the cleavage of the targeted HPRT genomic sequence by theCRISPR/Cas9-NLS complex transduced by FSD5, in the absence (“Notemplate”) or presence (“+500 ng”) of a long linear DNA template (1631bp). Thin dashed arrows indicate the bands corresponding to the targetgene, and thick solid arrows indicate the bands corresponding to theCRISPR/Cas9-NLS-mediated cleavage products of this target gene, whichindicate the successful transduction of fully functional genome editingcomplexes in the presence and absence of the DNA template. The numericalvalue in italics at the bottom of each lane is the sum of the relativesignals (%) of only the two cleavage product bands (thick solid arrows).FIG. 51J shows the results of a PCR-amplification of the samples of FIG.51I, using primers designed to amplify across the genomic cleavage site.Genomic insertion of the long DNA template sequence is visible by thepresence of a larger band in the “+500 ng” (faint) and “+1000 ng”(darker) lanes—see arrow in FIG. 51J.

FIGS. 51K and 51L show the results of the cleavage of the targetedgenomic DNMT1 DNA sequence with a CRISPR/Cpf1-NLS genome editing complexin the absence (“− ctrl”) or presence of the shuttle agent FSD18 in HeLa(FIG. 51K) and NK cells (FIG. 51L), after PCR-amplification andseparation by agarose gel electrophoresis. Thin dashed arrows indicatethe bands corresponding to the target gene, and thick solid arrowsindicate the bands corresponding to the CRISPR/Cpf1-NLS-mediatedcleavage products of this target gene, which indicate the successfultransduction of fully functional genome editing complexes. An imagingsoftware was used to quantify the relative signal intensities of each ofthe different bands directly on gels. The sum of all the bands in agiven lane corresponds to 100% of the signal, and the numerical value initalics at the bottom of each lane is the sum of the relative signals(%) of only the two cleavage product bands (thick solid arrows). Nogenomic DNA cleavage was observed using the lipofectamine-basedtransfection reagent CRISPRMAX™ to transduce the CRISPR/Cpf1-NLS genomeediting complex.

FIGS. 52A-52E show the results of the cleavage of a targeted genomic B2MDNA sequence with the CRISPR/Cas9-NLS and the crRNA/tracrRNA, or withthe CRISPR/Cpf1-NLS and a single guide RNA in the absence (“− ctrl”) orpresence of the shuttle agents FSD10, FSD18, FSD19, FSD21, FSD23 orFSD43 used at different concentrations, exposure times, and in differenttypes of cells: THP-1 (FIG. 52A); NK (FIG. 52B-D) and HeLa (FIG. 52E),after separation by agarose gel electrophoresis. Cells were incubatedwith CRISPR/Cpf1 complexes and FSD shuttle agents at the indicated timesand concentrations. Thin dashed arrows indicate the bands correspondingto the target gene, and thicker solid arrows indicate the bandscorresponding to the CRISPR system-mediated cleavage products of thistarget gene, which indicate the successful transduction of fullyfunctional CRISPR/Cas9-NLS genome editing complexes. An imaging softwarewas used to quantify the relative signal intensities of each of thedifferent bands directly on gels. The sum of all the bands in a givenlane corresponds to 100% of the signal, and the numerical value initalics at the bottom of each lane is the sum of the relative signals(%) of only the two cleavage product bands (thick solid arrows).

FIGS. 52F-52I show the results of the cleavage of a targeted genomicGSK3 (FIG. 52F), CBLB (FIG. 52G) and DNMT1 (FIG. 52H-I) DNA sequencewith the CRISPR/Cpf1-NLS and a single guide crRNA in the absence (“−ctrl”) or presence of the shuttle agents FSD10, FSD18, FSD19 or FSD23used at different concentrations, exposure times, and in different typesof cells: NK (FIG. 52F-G); THP-1 (FIG. 52H) and primary myoblasts (FIG.52I), after separation by agarose gel electrophoresis. Cells wereincubated with CRISPR/Cpf1 complexes and FSD at the indicated times andconcentrations. Thin dashed arrows indicate the bands corresponding tothe target gene, and thicker solid arrows indicate the bandscorresponding to the CRISPR system-mediated cleavage products of thesetarget genes, which indicate the successful transduction of fullyfunctional CRISPR/Cpf1-NLS genome editing complexes.

FIGS. 52J-52N show the results of the cleavage of a targeted genomicNKG2A DNA sequence with the CRISPR/Cpf1-NLS and a single guide crRNA inthe absence (“− ctrl”) or presence of the shuttle agents FSD10, FSD21,FSD22 or FSD23 used at different concentrations, exposure times, and inNK cells (FIG. 52F-52G) and NK-92 cells (FIG. 52H), after separation byagarose gel electrophoresis. Cells were incubated with CRISPR/Cpf1complexes for the indicated incubation times and concentrations. Thindashed arrows indicate the bands corresponding to the target gene, andthicker solid arrows indicate the bands corresponding to the CRISPRsystem-mediated cleavage products of this target gene, which indicatethe successful transduction of fully functional CRISPR/Cpf1-NLS genomeediting complexes.

FIGS. 53A-53C show the results of the cleavage of the targeted genomicHPRT, DNMT1 and B2M DNA sequences with CRISPR systems. The two genomicHPRT and DNMT1 (FIG. 53A) DNA sequences or two DNA loci on the genomicB2M exon 2 (FIG. 53B) were targeted in HeLa cells using CRISPR/Cas9-NLSand CRISPR/Cpf1-NLS genome editing complexes designed for thosepurposes, which were transduced using the shuttle agent peptide FSD18.The two DNA loci on the genomic B2M exon 2 (FIG. 53C) were targeted inNK cells with the CRISPR/Cpf1-NLS and single guide crRNA-1, crRNA-2 orboth, in the absence (“− ctrl”) or presence of the shuttle agents FSD10,FSD21 or FSD23 used at different concentrations, exposure times, afterseparation by agarose gel electrophoresis. Thin dashed arrows indicatethe bands corresponding to the target gene, and thicker solid arrowsindicate the bands corresponding to the CRISPR/Cpf1-mediated cleavageproducts in presence of the crRNA-1 or crRNA2 or both (crRNA1+2) for theB2M exon 2, which indicate the successful transduction of fullyfunctional CRISPR/Cpf1-NLS genome editing complexes.

FIGS. 54A-54D show the results of flow cytometry assays in which T cellswere treated with increasing concentrations of the shuttle agent FSD21(8 μM in FIG. 54B, 10 μM in FIG. 54C, and 12 μM in FIG. 54D), 1.33 μM ofCRISPR/Cpf1-NLS system and 2 μM of single guide crRNA targeting a B2Mgenomic DNA sequence. HLA-positive and HLA-negative (B2M knock-out)cells were identified 72 hours after treatment by using an APC-labeledMouse Anti-Human HLA-ABC antibody. Left-shifted cell populationsindicated successful inactivation of cell surface HLA receptors,resulting from inactivation of the B2M gene.

FIGS. 55A-55D show the results of flow cytometry assays in which T cellswere treated with increasing concentrations of the shuttle agent FSD18(8 μM in FIG. 55B, 10 μM in FIG. 55C, and 12 μM in FIG. 55D), 1.33 μM ofCRISPR/Cpf1-NLS system and 2 μM of single guide crRNA targeting a B2MDNA sequence. HLA-positive and HLA-negative (B2M knock-out) cells wereidentified 72 hours after treatment by using an APC-labeled MouseAnti-Human HLA-ABC antibody. Left-shifted cell populations indicatedsuccessful inactivation of cell surface HLA receptors, resulting frominactivation of the B2M gene.

FIGS. 56A-56E show the results of flow cytometry assays in which THP-1cells were treated (FIG. 56B-56E) or not (“untreated”, FIG. 56A) with amixture of 1.33 μM of CRISPR/Cpf1-NLS system, 2 μM of one or three guidecrRNA each targeting different sites within the B2M genomic DNA sequenceand 3 μM of FSD18. HLA-positive and HLA-negative (B2M knock-out) cellswere identified 48 hours after treatment by using an APC-labeled MouseAnti-Human HLA-ABC antibody in untreated cells (FIG. 56A), crRNA Etreated cells (FIG. 56B), crRNA G treated cells (FIG. 56C), crRNA Jtreated cells (FIG. 56D) and crRNA E+G+J treated cells (FIG. 56E).Left-shifted cell populations indicated successful inactivation of cellsurface HLA receptors, resulting from inactivation of the B2M gene.

FIGS. 57A and 57B show the results of experiments in which NK-92 cellswere genome-edited to determine whether inactivation of the endogenousNKG2A gene could increase their ability to kill target HeLa cells.Briefly, NK-92 cells were transduced with a CRISPR/Cpf1-NLS genomeediting complex designed to cleave the NKG2A gene using the shuttleagent peptide FSD23. After transduction, NK-92 cells were immunolabelledwith a phycoerythrin (PE)-labelled anti-NKG2A antibody and then analyzedby flow cytometry as shown in FIG. 57A, to verify successfulinactivation of NKG2A. As controls, unlabelled wild-type NK-92 cells(“unlabelled WT cells”) had no antibody signal, and labelled wild-typeNK-92 cells (“labelled WT cells”) had full immunolabelling signal. ForNKG2A-KO NK-92 cells, two cell populations (peaks) were observed: onewith a complete knock-out of NKG2A receptor expression on the cellsurface (“Complete NKG2A KO cells”), and the other with a partial lackof expression (“Partial NKG2A KO cells”). FIG. 57B shows the results ofcytotoxicity assays in which target HeLa cells previously loaded with anintracellular fluorescent dye (calcein) were exposed to either wild-type(solid line) or genome edited NKG2A-KO (dotted line) effector NK-92cells, at different Effector:Target ratios (E:T ratio). Cytotoxicity wasevaluated by the relative release of intracellular calcein into theextracellular space resulting from disruption of the cell membranes ofthe target HeLa cells (% cell lysis”).

FIGS. 58A-58F show the results of flow cytometry assays in which T cellswere treated (FIG. 58A-58F) or not (“untreated”, FIG. 58A) with amixture of GFP-NLS, a CRISPR/Cpf1-NLS complex designed to cleave the B2Mgenomic DNA sequence, and the shuttle agent FSD18. FIGS. 58B and 58Cshow the results of a two-dimensional flow cytometry analysis 5 h aftertreatment based on GFP fluorescence (x-axis) and cell surface HLAexpression (y-axis). Fluorescence-activated cell sorting (FIG. 58D) ofcells based on their GFP-fluorescence into a GFP-negative fraction (FIG.58E) and a GFP-positive fraction (FIG. 58F) resulted in an increase inthe proportion of genome-edited (HLA-negative) cells to 29.7% in theGFP-positive fraction (FIG. 58F). Each cell fraction was then subjectedto a standard T7E1 cleavage assay followed by agarose gelelectrophoresis to evaluate the effectiveness of the genome editing. Theresults are shown in FIG. 58G, wherein thin dashed arrows indicate thebands corresponding to the target gene, and thicker solid arrowsindicate the bands corresponding to the CRISPR system-mediated cleavageproducts of this target gene. The values at the bottom of lanes 2, 3 and4 correspond to the relative signal intensities (%) of the cleavageproduct bands (solid arrows) in that lane.

FIGS. 59A-59E show the results of flow cytometry assays in which T cellswere treated (FIG. 59C-59E) or not (“untreated”, FIGS. 59A and 59B) witha mixture GFP-NLS, a CRISPR/Cpf1-NLS complex designed to cleave theendogenous B2M gene, and the shuttle agent FSD18. FIGS. 59A and 59B,show the results of “untreated” negative control cells not exposed tothe peptide shuttle agent, GFP, nor CRISPR/Cpf1, which were analyzed byflow cytometry for GFP fluorescence (FIG. 59A) and cell surface HLAexpression (FIG. 59B). T cells were co-transduced with both GFP-NLS andCRISPR/Cpf1 via the peptide FSD18, and the resulting GFP fluorescencedistribution is shown in FIG. 59C. The two gates shown in FIG. 59Cindicate the fraction of cells that were considered to be GFP-positive(“GFP+”; 93.2%; solid gate) and the sub-fraction of cells that wereconsidered as exhibiting high GFP fluorescence (“GFP high”; 33.1%;dotted gate). Fluorescence-activated cell sorting was performed toquantify the level of cell surface HLA expression in cells considered tobe GFP-positive (FIG. 59D) as compared to cells considered as exhibitinghigh GFP fluorescence (FIG. 59E). The above co-transduction experimentwas repeated using 12 μM or 15 μM FSD18, followed byfluorescence-activated cell sorting into GFP-positive and GFP-negativecell fractions. Each fraction was subjected to a standard T7E1 cleavageassay, and the different samples were subjected to agarose gelelectrophoresis.

FIG. 60 shows the results of a flow cytometry analysis in which T cellswere subjected to a first transduction (FIG. 60B) or not (“untreated”,FIG. 60A) with the cargo GFP-NLS using the peptide FSD18. The firsttransduction resulted in a GFP-NLS transduction efficiency of 55.4%(FIG. 60B). Fluorescence-activated cell sorting was performed to isolateGFP-negative cells (FIG. 60C) following the first transduction, and thisGFP-negative cell population was subjected to a second transduction withGFP-NLS using the peptide FSD18. The results from this secondtransduction are shown in FIG. 60D, in which the GFP-NLS transductionefficiency was found to be 60.6%.

SEQUENCE LISTING

This application contains a Sequence Listing in computer readable formcreated Oct. 10, 2017 having a size of about 5 MB. The computer readableform is incorporated herein by reference.

SEQ ID NO: Description 1 CM18 2 Diphtheria toxin T domain (DT) 3 GALA 4PEA 5 INF-7 6 LAH4 7 HGP 8 H5WYG 9 HA2 10 EB1 11 VSVG 12 Pseudomonastoxin 13 Melittin 14 KALA 15 JST-1 16 SP 17 TAT 18 Penetratin(Antennapedia) 19 pVEC 20 M918 21 Pep-1 22 Pep-2 23 Xentry 24 Argininestretch 25 Transportan 26 SynB1 27 SynB3 28 E1a 29 SV40 T-Ag 30 c-myc 31Op-T-NLS 32 Vp3 33 Nucleoplasmin 34 Histone 2B NLS 35 Xenopus N1 36 PARR37 PDX-1 38 QKI-5 39 HCDA 40 H2B 41 v-Rel 42 Amida 43 RanBP3 44 Pho4p 45LEF-1 46 TCF-1 47 BDV-P 48 TR2 49 SOX9 50 Max 51 Mitochondrial signalsequence from Tim9 52 Mitochondrial signal sequence from Yeastcytochrome c oxidase subunit IV 53 Mitochondrial signal sequence from18S rRNA 54 Peroxisome signal sequence—PTS1 55 Nucleolar signal sequencefrom BIRC5 56 Nucleolar signal sequence from RECQL4 57 CM18-TAT 58CM18-Penetratin 59 His-CM18-TAT 60 GFP 61 TAT-GFP 62 GFP-NLS 63C(LLKK)3C 64 G(LLKK)3G 65 PTD4 66 TAT-CM18 67 TAT-KALA 68 His-CM18-PTD469 His-CM18-9Arg 70 His-CM18-Transportan 71 His-LAH4-PTD4 72His-C(LLKK)3C-PTD4 73 mCherryTM-NLS 74 Cas9-NLS 75 crRNA (Example 13.3)76 tracrRNA (Example 13.3) 77 Feldan tracrRNA (Example 13.5, 13.6) 78PPIB crRNA (Example 13.5) 79 Dharmacon tracrRNA (Example 13.5) 80HOXB4-WT 81 His-PTD4 82 PTD4-KALA 83 9Arg-KALA 84 Pep1-KALA 85Xentry-KALA 86 SynB3-KALA 87 VSVG-PTD4 88 EB1-PTD4 89 JST-PTD4 90CM18-PTD4 91 6Cys-CM18-PTD4 92 CM18-L1-PTD4 93 CM18-L2-PTD4 94CM18-L3-PTD4 95 His-CM18-TAT 96 His-CM18-PTD4-6Cys 97 3His-CM18-PTD4 9812His-CM18-PTD4 99 HA-CM18-PTD4 100 3HA-CM18-PTD4 101 CM18-His-PTD4 102His-CM18-PTD4-His 103 HPRT crRNA (Example 13.6) 104-129 FSD1-FSD26130-137 FSN1-FSN8 138-153 FSD27-FSD42 154 Short DNA template 155Cpf1-NLS 156 GFP coding DNA template 157 DNMT1 crRNA 158 LKLWXRXLKXXXXGmotif 159 RRXXAKXA motif 160 B2M crRNA (Cas9) 161 B2M exon 2 crRNA-1(Cpf1) 162 B2M exon 2 crRNA-2 (Cpf1) 163 CBLB crRNA 164 GSK3 crRNA 165NKG2A crRNA 166 B2M crRNA-E 167 B2M crRNA-J 168 B2M crRNA-G 169-242FSD43-FSD116 243-10 242 Computer-generated peptide variants that respectdesign parameters described herein

DETAILED DESCRIPTION

Large-scale screening efforts led to the discovery that domain-basedpeptide shuttle agents, comprising an endosome leakage domain (ELD)operably linked to a cell penetrating domain (CPD), and optionally oneor more histidine-rich domains, can increase the transduction efficiencyof an independent polypeptide cargo in eukaryotic cells, such that thecargo gains access to the cytosol/nuclear compartment (e.g., seeExamples 1-15). Conversely, the above screening efforts also revealedsome peptides having no or low polypeptide cargo transduction power,excessive toxicity, and/or other undesirable properties (e.g., poorsolubility and/or stability).

Based on these empirical data (both positive and negative), the aminoacid sequences and properties of successful, less successful, and failedpeptides were compared in order to better understand the physicochemicalproperties common to the more successful shuttle agents. This comparisoninvolved two main approaches: First, manually stratifying the differentscreened peptides according to their transduction performance, based onour complied biological characterization data; and second, a moresimplified “transduction score” approach, which considered only thetransduction efficiency and cellular toxicity of the different peptides,for a given polypeptide cargo and cell line.

For manual stratification, the screened peptides were evaluatedindividually according to their transduction performance, with dueconsideration to, for example: their solubility/stability/ease ofsynthesis; their ability to facilitate escape of endosomally-trappedcalcein (e.g., see Example 2); their ability to deliver one or moretypes of independent polypeptide cargos intracellularly, as evaluated byflow cytometry (e.g., see Examples 3-6 and 8-15) in different types ofcells and cell lines (e.g., primary, immortalized, adherent, suspension,etc.) as well as under different transduction protocols; their abilityto deliver polypeptide cargos to the cytosol and/or nucleus, asevaluated by fluorescence microscopy (e.g., for fluorescently labelledcargos), increased transcriptional activity (e.g., for transcriptionfactor cargos), or genome editing capabilities (e.g., for nucleasecargos or genome-editing complexes such as CRISPR/Cas9 or CRISPR/Cpf1)(e.g., see Examples 3-6 and 8-15), and toxicity towards different typesof cells and cell lines (e.g., primary, immortalized, adherent,suspension, etc.), under different transduction protocols.

For the “transduction score” approach, each peptide was assigned a scorecorresponding to a given cell line and fluorescently-labelledpolypeptide cargo, which combines both transduction efficiency andcellular toxicity data into a single numerical value. The transductionscores were calculated by simply multiplying the highest percentagetransduction efficiency observed by flow cytometry for a given peptide,cargo and cell type by the percentage cellular viability for the peptidein the tested cell line. The peptides were then sorted according totheir transduction scores as a screening tool to stratify peptides assuccessful, less successful, or failed shuttle agents.

The above-mentioned manual curation and “transduction score”-basedanalyses revealed a number of parameters that are generally shared bysuccessful domain-based shuttle agents (e.g. see Example A). Theseparameters were then successfully used to manually design new peptideshuttle agents having polypeptide cargo transduction activity, whichlack and/or are not based on known putative CPDs and/or ELDs (e.g., seeExample B). Furthermore, it was observed that peptides satisfying themost number of design parameters had generally the highest transductionscores, while peptides satisfying the least number of design parametershad generally the lowest transduction scores.

The design parameters described herein were further validated by testinga plurality of synthetic peptides whose amino acid sequences weregenerated using a machine learning algorithm (e.g., see Example C.1),the algorithm having been “trained” using transduction efficiency andcellular toxicity data of domain-based peptides (but not the designparameters described herein). Interestingly, the peptides generated bythe machine learning algorithm demonstrating the highest transductionscores were generally peptides that satisfied all of the designparameters described herein, thereby substantiating their use inactively designing and/or predicting the transduction activity of newpeptide shuttle agents (e.g., tailored to particular polypeptide cargosand/or types of cells). Furthermore, computer-assisted random peptidesequence generation followed by descriptors filtering was used togenerate a list of 10 000 peptide variants that respect nearly all ofthe design parameters described herein (see Example C.2).

Rationally-designed peptide shuttle agents are shown herein tofacilitate escape of an endosomally trapped fluorescent dye, suggestingendosomolytic activity (e.g., see Example D). Furthermore, the abilityof rationally-designed peptide shuttle agents to transduce a variety ofpolypeptide cargos (e.g., fluorescent proteins, transcription factors,antibodies, as well as entire CRISPR-associated genome editingcomplexes, with or without a DNA template) in a variety of differentcell types (both adherent and suspension) is also shown herein (e.g.,see Examples E-G).

Rational Design Parameters and Peptide Shuttle Agents

In some aspects, the present description relates to a method fordelivering a polypeptide cargo from an extracellular space to thecytosol and/or nucleus of a target eukaryotic cell. The method comprisescontacting the target eukaryotic cell with the polypeptide cargo in thepresence of a shuttle agent at a concentration sufficient to increasethe transduction efficiency of said polypeptide cargo, as compared to inthe absence of the shuttle agent. In some aspects, the shuttle agentrelates to a peptide that satisfies one or more of the followingparameters.

(1) In some embodiments, the shuttle agent is a peptide at least 20amino acids in length. For example, the peptide may comprise a minimumlength of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acidresidues, and a maximum length of 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 60, 65, 70, 75, 80, 85,90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 aminoacid residues. In some embodiments, shorter peptides (e.g., in the 20-50amino acid range) may be particularly advantageous because they may bemore easily synthesized and purified by chemical synthesis approaches,which may be more suitable for clinical use (as opposed to recombinantproteins that must be purified from cellular expression systems). Whilenumbers and ranges in the present description are often listed asmultiples of 5, the present description should not be so limited. Forexample, the maximum length described herein should be understood asalso encompassing a length of 56, 57, 58 . . . 61, 62, etc., in thepresent description, and that their non-listing herein is only for thesake of brevity. The same reasoning applies to the % of identitieslisted herein.(2) In some embodiments, the peptide shuttle agent comprises anamphipathic alpha-helical motif. As used herein, the expression“alpha-helical motif” or “alpha-helix”, unless otherwise specified,refers to a right-handed coiled or spiral conformation (helix) havingangle of rotation between consecutive amino acids of 100 degrees and/oran alpha-helix having 3.6 residues per turn. As used herein, theexpression “comprises an alpha-helical motif” or “an amphipathicalpha-helical motif” and the like, refers to the three-dimensionalconformation that a peptide (or segment of a peptide) of the presentdescription is predicted to adopt when in a biological setting based onthe peptide's primary amino acid sequence, regardless of whether thepeptide actually adopts that conformation when used in cells as ashuttle agent. Furthermore, the peptides of the present description maycomprise one or more alpha-helical motifs in different locations of thepeptide. For example, the shuttle agent FSD5 is predicted to adopt analpha-helix over the entirety of its length (see FIG. 49C), while theshuttle agent FSD18 is predicted to comprise two separate alpha-helicestowards the N and C terminal regions of the peptide (see FIG. 49D). Insome embodiments, the shuttle agents of the present description are notpredicted to comprise a beta-sheet motif, for example as shown in FIGS.49E and 49F. Methods of predicting the presence of alpha-helices andbeta-sheets in proteins and peptides are well known in the art. Forexample, one such method is based on 3D modeling using PEP-FOLD™, anonline resource for de novo peptide structure prediction(http://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD/) (Lamiableet al., 2016; Shen et al., 2014; Thévenet et al., 2012). Other methodsof predicting the presence of alpha-helices in peptides and protein areknown and readily available to the skilled person.

As used herein, the expression “amphipathic” refers to a peptide thatpossesses both hydrophobic and hydrophilic elements (e.g., based on theside chains of the amino acids that comprise the peptide). For example,the expression “amphipathic alpha helix” or “amphipathic alpha-helicalmotif” refers to a peptide predicted to adopt an alpha-helical motifhaving a non-polar hydrophobic face and a polar hydrophilic face, basedon the properties of the side chains of the amino acids that form thehelix.

(3) In some embodiments, peptide shuttle agents of the presentdescription comprise an amphipathic alpha-helical motif having apositively-charged hydrophilic outer face, such as one that is rich in Rand/or K residues. As used herein, the expression “positively-chargedhydrophilic outer face” refers to the presence of at least three lysine(K) and/or arginine (R) residues clustered to one side of theamphipathic alpha-helical motif, based on alpha-helical wheel projection(e.g., see FIG. 49A, left panel). Such helical wheel projections may beprepared using a variety of programs, such as the online helical wheelprojection tool available at:http://rzlab.ucr.edu/scripts/wheel/wheel.cgi. In some embodiments, theamphipathic alpha-helical motif may comprise a positively-chargedhydrophilic outer face that comprises: (a) at least two, three, or fouradjacent positively-charged K and/or R residues upon helical wheelprojection; and/or (b) a segment of six adjacent residues comprisingthree to five K and/or R residues upon helical wheel projection, basedon an alpha helix having angle of rotation between consecutive aminoacids of 100 degrees and/or an alpha-helix having 3.6 residues per turn.

In some embodiments, peptide shuttle agents of the present descriptioncomprise an amphipathic alpha-helical motif comprising a hydrophobicouter face, the hydrophobic outer face comprising: (a) at least twoadjacent L residues upon helical wheel projection; and/or (b) a segmentof ten adjacent residues comprising at least five hydrophobic residuesselected from: L, I, F, V, W, and M, upon helical wheel projection,based on an alpha helix having angle of rotation between consecutiveamino acids of 100 degrees and/or an alpha-helix having 3.6 residues perturn.

(4) In some embodiments, peptide shuttle agents of the presentdescription comprise an amphipathic alpha-helical motif having a highlyhydrophobic core composed of spatially adjacent highly hydrophobicresidues (e.g., L, I, F, V, W, and/or M). In some embodiments, thehighly hydrophobic core may consist of spatially adjacent L, I, F, V, W,and/or M amino acids representing 12 to 50% of the amino acids of thepeptide, calculated while excluding any histidine-rich domains (seebelow), based on an open cylindrical representation of the alpha-helixhaving 3.6 residues per turn, as shown for example in FIG. 49A, rightpanel. In some embodiments, the highly hydrophobic core may consist ofspatially adjacent L, I, F, V, W, and/or M amino acids representing from12.5%, 13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%,18.5%, 19%, 19.5%, or 20%, to 25%, 30%, 35%, 40%, or 45% of the aminoacids of the peptide. More particularly, highly hydrophobic coreparameter may be calculated by first arranging the amino acids of thepeptide in an opened cylindrical representation, and then delineating anarea of contiguous highly hydrophobic residues (L, I, F, V, W, M), asshown in FIG. 49A, right panel. The number of highly hydrophobicresidues comprised in this delineated highly hydrophobic core is thendivided by the total amino acid length of the peptide, excluding anyhistidine-rich domains (e.g., N- and/or C-terminal histidine-richdomains). For example, for the peptide shown in FIG. 49A, there are 8residues in the delineated highly hydrophobic core, and 25 totalresidues in the peptide (excluding the terminal 12 histidines). Thus,the highly hydrophobic core is 32% (8/25).(5) Hydrophobic moment relates to a measure of the amphiphilicity of ahelix, peptide, or part thereof, calculated from the vector sum of thehydrophobicities of the side chains of the amino acids (Eisenberg atal., 1982). An online tool for calculating the hydrophobic moment of apolypeptide is available from:http://rzlab.ucr.edu/scripts/wheel/wheel.cgi. A high hydrophobic momentindicates strong amphiphilicity, while a low hydrophobic momentindicates poor amphiphilicity. In some embodiments, peptide shuttleagents of the present description may consist of or comprise a peptideor alpha-helical domain having have a hydrophobic moment (μ) of 3.5 to11. In some embodiments, the shuttle agent may be a peptide comprisingan amphipathic alpha-helical motif having a hydrophobic moment between alower limit of 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5,4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9,6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, and an upperlimit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5,10.6, 10.7, 10.8, 10.9, or 11.0. In some embodiments, the shuttle agentmay be a peptide having a hydrophobic moment between a lower limit of4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3,5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7,6.8, 6.9, 7.0, and an upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0,10.1, 10.2, 10.3, 10.4, or 10.5. In some embodiments, the hydrophobicmoment is calculated excluding any histidine-rich domains that may bepresent in the peptide.(6) In some embodiments, peptide shuttle agents of the presentdescription may have a predicted net charge of at least +4 atphysiological pH, calculated from the side chains of K, R, D, and Eresidues. For example, the net charge of the peptide may be at least +5,+6, +7, at least +8, at least +9, at least +10, at least +11, at least+12, at least +13, at least +14, or at least +15 at physiological pH.These positive charges are generally conferred by the greater presenceof positively-charged lysine and/or arginine residues, as opposed tonegatively charged aspartate and/or glutamate residues.(7) In some embodiments, peptide shuttle agents of the presentdescription may have a predicted isoelectric point (pI) of 8 to 13,preferably from 10 to 13. Programs and methods for calculating and/ormeasuring the isoelectric point of a peptide or protein are known in theart. For example, pI may be calculated using the Prot Param softwareavailable at: http://web.expasy.org/protparam/(8) In some embodiments, peptide shuttle agents of the presentdescription may be composed of 35 to 65% of hydrophobic residues (A, C,G, I, L, M, F, P, W, Y, V). In particular embodiments, the peptideshuttle agents may be composed of 36% to 64%, 37% to 63%, 38% to 62%,39% to 61%, or 40% to 60% of any combination of the amino acids: A, C,G, I, L, M, F, P, W, Y, and V.(9) In some embodiments, peptide shuttle agents of the presentdescription may be composed of 0 to 30% of neutral hydrophilic residues(N, Q, S, T). In particular embodiments, the peptide shuttle agents maybe composed of 1% to 29%, 2% to 28%, 3% to 27%, 4% to 26%, 5% to 25%, 6%to 24%, 7% to 23%, 8% to 22%, 9% to 21%, or 10% to 20% of anycombination of the amino acids: N, Q, S, and T.(10) In some embodiments, peptide shuttle agents of the presentdescription may be composed of 35 to 85% of the amino adds A, L, Kand/or R. In particular embodiments, the peptide shuttle agents may becomposed of 36% to 80%, 37% to 75%, 38% to 70%, 39% to 65%, or 40% to60% of any combination of the amino adds: A, L, K, or R.(11) In some embodiments, peptide shuttle agents of the presentdescription may be composed of 15 to 45% of the amino acids A and/or L,provided there being at least 5% of L in the peptide. In particularembodiments, the peptide shuttle agents may be composed of 15% to 40%,20% to 40%, 20 to 35%, or 20 to 30% of any combination of the aminoacids: A and L, provided there being at least 5% of L in the peptide.(12) In some embodiments, peptide shuttle agents of the presentdescription may be composed of 20 to 45% of the amino acids K and/or R.In particular embodiments, the peptide shuttle agents may be composed of20% to 40%, 20 to 35%, or 20 to 30% of any combination of the aminoacids: K and R.(13) In some embodiments, peptide shuttle agents of the presentdescription may be composed of 0 to 10% of the amino acids D and/or E.In particular embodiments, the peptide shuttle agents may be composed of5 to 10% of any combination of the amino acids: D and E.(14) In some embodiments, the absolute difference between the percentageof A and/or L and the percentage of K and/or R in the peptide shuttleagent may be less than or equal to 10%. In particular embodiments, theabsolute difference between the percentage of A and/or L and thepercentage of K and/or R in the peptide shuttle agent may be less thanor equal to 9%, 8%, 7%, 6%, or 5%.(15) In some embodiments, peptide shuttle agents of the presentdescription may be composed of 10% to 45% of the amino acids Q, Y, W, P,I, S, G, V, F, E, D, C, M, N, T, or H (i.e., not A, L, K, or R). Inparticular embodiments, the peptide shuttle agents may be composed of 15to 40%, 20% to 35%, or 20% to 30% of any combination of the amino acids:Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H.

In some embodiments, peptide shuttle agents of the present descriptionrespect at least one, at least two, at least three, at least four, atleast five, at least six, at least seven, at least eight, at least nine,at least ten, at least eleven, at least twelve, at leave thirteen, atleast fourteen, or all of parameters (1) to (15) described herein. Inparticular embodiments, peptide shuttle agents of the presentdescription respect all of parameters (1) to (3), and at least six, atleast seven, at least eight, at least nine, at least ten, at leasteleven, or all of parameters (4) to (15) described herein.

In some embodiments, where a peptide shuttle agent of the presentdescription comprises only one histidine-rich domain, the residues ofthe one histidine-rich domain may be included in thecalculation/assessment of parameters (1) to (15) described herein. Insome embodiments, where a peptide shuttle agent of the presentdescription comprises more than one histidine-rich domain, only theresidues of one of the histidine-rich domains may be included in thecalculation/assessment of parameters (1) to (15) described herein. Forexample, where a peptide shuttle agent of the present descriptioncomprises two histidine-rich domains: a first histidine-rich domaintowards the N terminus, and a second histidine-rich domain towards the Cterminus, only the first histidine-rich domain may be included in thecalculation/assessment of parameters (1) to (15) described herein.

In some embodiments, a machine-learning or computer-assisted designapproach may be implemented to generate peptides that respect one ormore of parameters (1) to (15) described herein. Some parameters, suchas parameters (1) and (5)-(15), may be more amenable to implementationin a computer-assisted design approach, while structural parameters,such as parameters (2), (3) and (4), may be more amenable to a manualdesign approach. Thus, in some embodiments, peptides that respect one ormore of parameters (1) to (15) may be generated by combiningcomputer-assisted and manual design approaches. For example, multiplesequence alignment analyses of a plurality of peptides shown herein (andothers) to function as effective shuttle agents revealed the presence ofsome consensus sequences—i.e., commonly found patterns of alternance ofhydrophobic, cationic, hydrophilic, alanine and glycine amino acids. Thepresence of these consensus sequences are likely to give rise tostructural parameters (2), (3) and (4) being respected (i.e.,amphipathic alpha-helix formation, a positively-charged face, and ahighly hydrophobic core of 12%-50%). Thus, these and other consensussequences may be employed in machine-learning and/or computer-assisteddesign approaches to generate peptides that respect one or of parameters(1)-(15).

Accordingly, in some embodiments, peptide shuttle agents describedherein may comprise or consist of the amino acid sequence of:

(a) [X1]-[X2]-[linker]-[X3]-[X4]  (Formula 1);

(b) [X1]-[X2]-[linker]-[X4]-[X3]  (Formula 2);

(c) [X2]-[X1]-[linker]-[X3]-[X4]  (Formula 3);

(d) [X2]-[X1]-[linker]-[X4]-[X3]  (Formula 4);

(e) [X3]-[X4]-[linker]-[X1]-[X2]  (Formula 5);

(f) [X3]-[X4]-[linker]-[X2]-[X1]  (Formula 6);

(g) [X4]-[X3]-[linker]-[X1]-[X2]  (Formula 7); or

(h) [X4]-[X3]-[linker]-[X2]-[X1]  (Formula 8),

wherein:

-   -   [X1] is selected from: 2[ϕ]−1[+]−2[ϕ]−1[ζ]−1[+]−;        2[ϕ]−1[+]−2[ϕ]−2[+]−; 1[+]−1[ϕ]−1[+]−2[ϕ]−1[ζ]−1[+]−; and        1[+]−1[ϕ]−1[+]−2[ϕ]−2[+]−;    -   [X2] is selected from: −2[ϕ]−1[+]−2[ϕ]−2−[ζ]−;        −2[ϕ]−1[+]−2[ϕ]−2[+]−; −2[ϕ]−1[+]−2[ϕ]−1[+]−1[ζ]−;        −2[ϕ]−1[+]−2[ϕ]−1[ζ]−1[+]−; −2[ϕ]−2[+]−1[ϕ]−2[+]−;        −2[ϕ]−2[+]−1[ϕ]−2[ζ]−; −2[ϕ]−2[+]−1[ϕ]−1[+]−1[ζ]−; and        −2[ϕ]−2[+]−1[ϕ]−1[ζ]−1[+]−;    -   [X3] is selected from: −4[+]−A−; −3[+]−G−A−; −3[+]−A−A−;        −2[+]−1[ϕ]−1[+]−A−; −2[+]−1[ϕ]−G−A−; −2[+]−1[ϕ]−A−A−; or        −2[+]−A−1[+]−A; −2[+]−A−G−A; −2[+]−A−A−A−; −1[ϕ]−3[+]−A−;        −1[ϕ]−2[+]−G−A−; −1[ϕ]−2[+]−A−A−; −1[ϕ]−1[+]−1[ϕ]−1[+]−A;        −1[ϕ]−1[+]−1[ϕ]−G−A; −1[ϕ]−1[+]−1[ϕ]−A−A−; 1[ϕ]−1[+]−A−1[+]−A;        −1[ϕ]−1[+]−A−G−A; −1[ϕ]−1[+]−A−A−A; −A−1[+]−A−1[+]−A;        −A−1[+]−A−G−A; and −A−1[+]−A−A−A;    -   [X4] is selected from: −1[ζ]−2A−1[+]−A; −1[ζ]−2A−2[+];        −1[+]−2A−1[+]−A; −1[ζ]−2A−1[+]−1[ζ]−A−1[+]; −1[ζ]−A−1[ϕ]−A−1[+];        −2[+]−A−2[+]; −2[+]−A−1[+]−A; −2[+]−A−1[+]−−1[ζ]−A−1[+];        −2[+]−1[ζ]−A−1[+]; −1[+]−1[ζ]−A−1[+]−A; −1[+]−1[ζ]−A−2[+];        −1[+]−1[ζ]−A−1[+]−1[ζ]−A−1[+]; −1[+]−2[ζ]−A−1[+];        −1[+]−2[ζ]−2[+]; −1[+]−2[ζ]−1[+]−A; −1[+]−2[ζ]−1[+]−1[ζ]−A−1[+];        −1[+]−2[ζ]−1[ζ]−A−1[+]; −3[ζ]−2[+]; −3[ζ]−1[+]−A;        −3[ζ]−1[+]−1[ζ]−A−1[+]; −1[ζ]−2A−1[+]−A; −1[ζ]−2A−2[+];        −1[ζ]−2A−1[+]−1[ζ]−A−1[+]; −2[+]−A−1[+]−A; −2[+]−1[ζ]−1[+]−A;        −1[+]−1[ζ]−A−1[+]−A; −1[+]−2A−1[+]−1[ζ]−A−1[+]; and        −1[ζ]−A−1[ζ]−A−1[+]; and    -   [linker] is selected from: -Gn-; −Sn-; −(GnSn)n-; −(GnSn)nGn-;        −(GnSn)nSn-; −(GnSn)nGn(GnSn)n-; and -(GnSn)nSn(GnSn)n-;        wherein: [ϕ] is an amino add which is Leu, Phe, Trp, Ile, Met,        Tyr, or Val; [+] is an amino acid which is: Lys or Arg; [ζ] is        an amino acid which is: Gln, Asn, Thr, or Ser; A is the a acid        Ala; G is the amino add Gly; S is the amino acid Ser, and n is        an integer from 1 to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1        to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10, 1 to 9, 1 to        8, 1 to 7, 1 to 6, 1 to 5, 1 to 1 to 4, or 1 to 3.

In some embodiments, peptide shuttle agents of the present descriptionmay comprise or consist of any one of the amino acid sequences of SEQ IDNOs: 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145, 148, 151,152, 169-242, and 243-10 242. In some embodiments, peptide shuttleagents of the present description may comprise the amino acid sequencemotifs of SEQ ID NOs: 158 and/or 159, which were found in each ofpeptides FSD5, FSD16, FSD18, FSD19, FSD20, FSD22, and FSD23. In someembodiments, peptide shuttle agents of the present description maycomprise the amino acid sequence motif of SEQ ID NO: 158 operably linkedto the amino acid sequence motif of SEQ ID NO: 159. In some embodiments,peptide shuttle agents of the present description may comprise orconsist of a peptide which is at least 50%, 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, or 95% identical to the amino acid sequence of any one ofSEQ ID NOs: 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142, 145,148, 151, 152, 169-242, and 243-10 242, or a functional variant of anyone of SEQ ID NOs: 104, 105, 107, 108, 110-131, 133-135, 138, 140, 142,145, 148, 151, 152, 169-242, and 243-10 242. As used herein, a“functional variant” refers to a peptide having polypeptide cargotransduction activity, which differs from the reference peptide by oneor more conservative amino add substitutions. As used herein, a“conservative amino acid substitution” is one in which one amino acidresidue is replaced with another amino acid residue having a similarside chain. Families of amino acid residues having similar side chainshave been well defined in the art, including basic side chains (e.g.,lysine, arginine, histidine), acidic side chains (e.g., aspartic acid,glutamic acid), uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains(e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan), beta-branched side chains (e.g., threonine,valine, isoleucine) and aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan, histidine).

In some embodiments, peptide shuttle agents of the present descriptionmay comprise or consist of the amino acid sequence of any one of SEQ IDNOs: 57-59, 66-72, or 82-102, or a functional variant thereof having atleast 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 79%, 80%, 81% 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, or 95% identity to any one of SEQ ID NOs: 57-59, 66-72,or 82-102. In some embodiments, peptide shuttle agents of the presentdescription do not comprise one or more of the amino acid sequences ofany one of SEQ ID NOs: 57-59, 66-72, or 82-102.

In some embodiments, shuttle agents of the present description maycomprise oligomers (e.g., dimers, trimers, etc.) of peptides describedherein. Such oligomers may be constructed by covalently binding the sameor different types of shuttle agent monomers (e.g., using disulfidebridges to link cysteine residues introduced into the monomersequences). In some embodiments, shuttle agents of the presentdescription may comprise an N-terminal and/or a C-terminal cysteineresidue.

Histidine-Rich Domains

In some embodiments, peptide shuttle agents of the present descriptionmay further comprise one or more histidine-rich domains. In someembodiments, the histidine-rich domain may be a stretch of at least 2,at least 3, at least 4, at least 5, or at least 6 amino acids comprisingat least 30%, at least 35%, at least 40%, at least 45%, at least 50%, atleast 55%, at least 60%, at least 65%, at least 70%, at least 75%, atleast 80%, at least 85%, or at least 90% histidine residues. In someembodiments, the histidine-rich domain may comprise at least 2, at least3, at least 4 at least 5, at least 6, at least 7, at least 8, or atleast 9 consecutive histidine residues. Without being bound by theory,the histidine-rich domain in the shuttle agent may act as a protonsponge in the endosome through protonation of their imidazole groupsunder acidic conditions of the endosomes, providing another mechanism ofendosomal membrane destabilization and thus further facilitating theability of endosomally-trapped cargos to gain access to the cytosol. Insome embodiments, the histidine-rich domain may be located at or towardsthe N and/or C terminus of the peptide shuttle agent.

Linkers

In some embodiments, peptide shuttle agents of the present descriptionmay comprise one or more suitable linkers (e.g., flexible polypeptidelinkers). In some embodiments, such linkers may separate two or moreamphipathic alpha-helical motifs (e.g., see the shuttle agent FSD18 inFIG. 49D). In some embodiments, linkers can be used to separate two moredomains (CPDs, ELDs, or histidine-rich domains) from one another. Insome embodiments, linkers may be formed by adding sequences of smallhydrophobic amino acids without rotatory potential (such as glycine) andpolar serine residues that confer stability and flexibility. Linkers maybe soft and allow the domains of the shuttle agents to move. In someembodiments, prolines may be avoided since they can add significantconformational rigidity. In some embodiments, the linkers may beserine/glycine-rich linkers (e.g., GGS, GGSGGGS, GGSGGGSGGGS, or thelike). In some embodiments, the use shuttle agents comprising a suitablelinker may be advantageous for delivering an independent polypeptidecargo to suspension cells, rather than to adherent cells. In someembodiments, the linker may comprise or consist of -Gn-; −Sn-;−(GnSn)n-; −(GnSn)nGn-; −(GnSn)nSn-; −(GnSn)nGn(GnSn)n- or-(GnSn)nSn(GnSn)n-, wherein G is the amino acid Gly; S is the amino acidSer; and n is an integer from 1 to 5.

Endosome Leakage Domains (ELDs)

In some aspects, peptide shuttle agents of the present description maycomprise an endosome leakage domain (ELD) for facilitating endosomeescape and access to the cytoplasmic compartment. As used herein, theexpression “endosome leakage domain” refers to a sequence of amino acidswhich confers the ability of endosomally-trapped macromolecules to gainaccess to the cytoplasmic compartment Without being bound by theory,endosome leakage domains are short sequences (often derived from viralor bacterial peptides), which are believed to induce destabilization ofthe endosomal membrane and liberation of the endosome contents into thecytoplasm. As used herein, the expression “endosomolytic peptide” isintended to refer to this general class of peptides having endosomalmembrane-destabilizing properties. Accordingly, in some embodiments,synthetic peptide or polypeptide-based shuttle agents of the presentdescription may comprise an ELD which is an endosomolytic peptide. Theactivity of such peptides may be assessed for example using the calceinendosome escape assays described in Example 2.

In some embodiments, the ELD may be a peptide that disrupts membranes atacidic pH, such as pH-dependent membrane active peptide (PMAP) or apH-dependent lytic peptide. For example, the peptides GALA and INF-7 areamphiphilic peptides that form alpha helixes when a drop in pH modifiesthe charge of the amino acids which they contain. More particularly,without being bound by theory, it is suggested that ELDs such as GALAinduce endosomal leakage by forming pores and flip-flop of membranelipids following conformational change due to a decrease in pH (Kakudo,Chaki et al., 2004, Li, Nicol et al., 2004). In contrast, it issuggested that ELDs such as INF-7 induce endosomal leakage byaccumulating in and destabilizing the endosomal membrane (El-Sayed,Futaki et al., 2009). Accordingly, in the course of endosome maturation,the concomitant decline in pH causes a change in the conformation of thepeptide and this destabilizes the endosome membrane leading to theliberation of the endosome contents. The same principle is thought toapply to the toxin A of Pseudomonas (Varkouhi, Scholte et al., 2011).Following a decline in pH, the conformation of the domain oftransiocation of the toxin changes, allowing its insertion into theendosome membrane where it forms pores (London 1992, O'Keefe 1992). Thiseventually favors endosome destabilization and translocation of thecomplex outside of the endosome. The above described ELDs areencompassed within the ELDs of the present description, as well as othermechanisms of endosome leakage whose mechanisms of action may be lesswell defined.

In some embodiments, the ELD may be an antimicrobial peptide (AMP) suchas a linear cationic alpha-helical antimicrobial peptide (AMP). Thesepeptides play a key role in the innate immune response due to theirability to strongly interact with bacterial membranes. Without beingbound by theory, these peptides are thought to assume a disordered statein aqueous solution, but adopt an alpha-helical secondary structure inhydrophobic environments. The latter conformation thought to contributeto their typical concentration-dependent membrane-disrupting properties.When accumulated in endosomes at certain concentrations, someantimicrobial peptides may induce endosomal leakage.

In some embodiments, the ELD may be an antimicrobial peptide (AMP) suchas Cecropin-A/Melittin hybrid (CM series) peptide. Such peptides arethought to be among the smallest and most effective AMP-derived peptideswith membrane-disrupting ability. Cecropins are a family ofantimicrobial peptides with membrane-perturbing abilities against bothGram-positive and Gram-negative bacteria. Cecropin A (CA), the firstidentified antibacterial peptide, is composed of 37 amino acids with alinear structure. Melittin (M), a peptide of 26 amino acids, is a cellmembrane lytic factor found in bee venom. Cecropin-melittin hybridpeptides have been shown to produce short efficient antibiotic peptideswithout cytotoxicity for eukaryotic cells (i.e., non-hemolytic), adesirable property in any antibacterial agent. These chimeric peptideswere constructed from various combinations of the hydrophilic N-terminaldomain of Cecropin A with the hydrophobic N-terminal domain of Melittin,and have been tested on bacterial model systems. Two 26-mers,CA(1-13)M(1-13) and CA(1-8) M(1-18) (Boman et al., 1989), have beenshown to demonstrate a wider spectrum and improved potency of naturalCecropin A without the cytotoxic effects of melittin.

In an effort to produce shorter CM series peptides, the authors ofAndreu et al., 1992 constructed hybrid peptides such as the 26-mer(CA(1-8)M(1-18)), and compared them with a 20-mer (CA(1-8)M(1-12)), a18-mer (CA(1-8)M(1-10)) and six 15-mers ((CA(1-7)M(1-8), CA(1-7)M(2-9),CA(1-7)M(3-10), CA(1-7)M(4-11), CA(1-7)M(5-12), and CA(1-7)M(6-13)). The20 and 18-mers maintained similar activity comparatively toCA(1-8)M(1-18). Among the six 15-mers, CA(1-7)M(1-8) showed lowantibacterial activity, but the other five showed similar antibioticpotency compared to the 26-mer without hemolytic effect. Accordingly, insome embodiments, synthetic peptide or polypeptide-based shuttle agentsof the present description may comprise an ELD which is or is from CMseries peptide variants, such as those described above.

In some embodiments, the ELD may be the CM series peptide CM18 composedof residues 1-7 of Cecropin-A (KWKLFKKIGAVLKVLTTG) fused to residues2-12 of Melittin (YGRKKRRQRRR), [C(1-7)M(2-12)]. When fused to the cellpenetrating peptide TAT, CM18 was shown to independently cross theplasma membrane and destabilize the endosomal membrane, allowing someendosomally-trapped cargos to be released to the cytosol (Salomone etal., 2012). However, the use of a CM18-TAT11 peptide fused to afluorophore (atto-633) in some of the authors' experiments, raisesuncertainty as to the contribution of the peptide versus thefluorophore, as the use of fluorophores themselves have been shown tocontribute to endosomolysis—e.g., via photochemical disruption of theendosomal membrane (Erazo-Oliveras et al., 2014).

In some embodiments, the ELD may be CM18 having the amino acid sequenceof SEQ ID NO: 1, or a variant thereof having at least 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 85%, 90%, 91%, 92%, 93%, 94%, or 95% identity to SEQ IDNO: 1 and having endosomolytic activity.

In some embodiments, the ELD may be a peptide derived from the Nterminus of the HA2 subunit of influenza hemagglutinin (HA), which mayalso cause endosomal membrane destabilization when accumulated in theendosome.

In some embodiments, synthetic peptide or polypeptide-based shuttleagents of the present description may comprise an ELD which is or isfrom an ELD set forth in Table I, or a variant thereof having endosomeescape activity and/or pH-dependent membrane disrupting activity.

TABLE I Examples of endosome leakage domains Name Amino acid sequenceSEQ ID NO: Reference(s) CM18 KWKLFKKIGAVLKVLTTG  1(Salomone, Cardarelli et al., 2012) Diphtheria toxin TVGSSLSCINLDWDVIRDKTKTKIESLKEHGPIKNK  2 (Uherek, Fominaya et al.,domain (DT) MSESPNKTVSEEKAKQYLEEFHQTALEHPELSEL 1998, Glover, Ng et al.,KTVIGINPVFAGANYMWAVNVAQVIDSETADNL 2009)EKTTAALSILPGIGSVMGIADGAVHHNTEEIVAQSI ALSSLMVAQAIPLVGELVDIGFAAYNFVESIINLFQVVHNSYNRPAYSPG GALA WEAALAEALAEALAEHLAEALAEALEALAA  3(Parente, Nir et al., 1990) (Li, Nicol et al., 2004) PEAVLAGNPAKHDLDIKPTVISHRLHFPEGGSLAALTA  4 (Fominaya and WelsHQACHLPLETFTRHRQPRGWEQLEQCGYPVQRL 1996) VALYLAARLSWNQVDQVIRNALASPGSGGDLGEAIREQPEQARLALT INF-7 GLFEAIEGFIENGWEGMIDGWYGC  5(El-Sayed, Futaki et al., 2009) LAH4 KKALLALALHHLAHLALHLALALKKA  6(Kichler, Mason et al., 2006) Kichler et al., 2003 HGPLLGRRGWEVLKYWWNLLQYWSQEL  7 Kwon et al., 2010 H5WYGGLFHAIAHFIHGGWHGLIHGWYG  8 (Midoux, Kichler et al., 1998) HA2GLFGAIAGFIENGWEGMIDGWYG  9 (Lorieau, Louis et al., 2010) EB1LIRLWSHLIHIWFQNRRLKWKKK 10 (Amand, Norden et al., 2012) VSVGKFTIVFPHNQKGNWKNVPSNYHYCP 11 (Schuster, Wu et al., 1999) PseudomonasEGGSLAALTAHQACHLPLETFTRHRQPRGWEQL 12 (Fominaya, Uherek et al., toxinEQCGYPVQRLVALYLAARLSWNQVDQVIRNALAS 1998)PGSGGDLGEAIREQPEQARLALTLAAAESERFVR QGTGNDEAGAANAD MelittinGIGAVLKVLTTGLPALISWIKRKRQQ 13 Tan, Chen et al., 2012) KALAWEAKLAKALAKALAKHLAKALAKALKACEA 14 (Wyman, Nicol et at, 1997) JST-1GLFEALLELLESLWELLLEA 15 (Gottschalk, Sparrow et al., 1996) C(LLKK)₃CCLLKKLLKKLLKKC 63 (Luan et al., 2014) G(LLKK)₃G GLLKKLLKKLLKKG 64(Luan et al., 2014)

In some embodiments, shuttle agents of the present description maycomprise one or more ELD or type of ELD. More particularly, they cancomprise at least 2, at least 3, at least 4, at least 5, or more ELDs.In some embodiments, the shuttle agents can comprise between 1 and 10ELDs, between 1 and 9 ELDs, between 1 and 8 ELDs, between 1 and 7 ELDs,between 1 and 6 ELDs, between 1 and 5 ELDs, between 1 and 4 ELDs,between 1 and 3 ELDs, etc.

In some embodiments, the order or placement of the ELD relative to theother domains (CPD, histidine-rich domains) within the shuttle agents ofthe present description may be varied provided the shuttling ability ofthe shuttle agent is retained.

In some embodiments, the ELD may be a variant or fragment of any onethose listed in Table I, and having endosomolytic activity. In someembodiments, the ELD may comprise or consist of the amino acid sequenceof any one of SEQ ID NOs: 1-15, 63, or 64, or a sequence which is atleast 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%,83%, 84%, 85%, 86%, 87%, 88%, 89%, 85%, 90%, 91%, 92%, 93%, 94%, or 95%identical to any one of SEQ ID NOs: 1-15, 63, or 64, and havingendosomolytic activity.

In some embodiments, shuttle agents of the present description do notcomprise one or more of the amino acid sequence of any one of SEQ IDNOs: 1-15, 63, or 64.

Cell Penetration Domains (CPDs)

In some aspects, the shuttle agents of the present description maycomprise a cell penetration domain (CPD). As used herein, the expression“cell penetration domain” refers to a sequence of amino acids whichconfers the ability of a macromolecule (e.g., peptide or protein)containing the CPD to be transduced into a cell.

In some embodiments, the CPD may be (or may be from) a cell-penetratingpeptide or the protein transduction domain of a cell-penetratingpeptide. Cell-penetrating peptides can serve as carriers to successfullydeliver a variety of cargos intracellularly (e.g., polynucleotides,polypeptides, small molecule compounds or other macromolecules/compoundsthat are otherwise membrane-impermeable). Cell-penetrating peptidesoften include short peptides rich in basic amino acids that, once fused(or otherwise operably linked) to a macromolecule, mediate itsinternalization inside cells (Shaw, Catchpole et al., 2008). The firstcell-penetrating peptide was identified by analyzing the cellpenetration ability of the HIV-1 trans-activator of transcription (Tat)protein (Green and Loewenstein 1988, Vives, Brodin et al., 1997). Thisprotein contains a short hydrophilic amino acid sequence, named “TAT”,which promotes its insertion within the plasma membrane and theformation of pores. Since this discovery, many other cell-penetratingpeptides have been described. In this regard, in some embodiments, theCPD can be a cell-penetrating peptide as listed in Table II, or avariant thereof having cell-penetrating activity.

TABLE II Examples of cell-penetrating peptides SEQ ID NameAmino acid sequence NO: Reference(s) SP AAVALLPAVLLALLAP 16(Mahlum, Mandal et al., 2007) TAT YGRKKRRQRRR 17(Green and Loewenstein 1988, Fawell, Seery et al., 1994, Vives,Brodin et al., 1997) Penetratin RQIKIWFQNRRMKWKK 18(Perez, Joliot et al., 1992) (Antennapedia) pVEC LLIILRRRIRKQAHAHSK 19(Elmquist, Lindgren et al., 2001) M918 MVTVLFRRLRIRRACGPPRVRV 20(El-Andaloussi, Johansson et al., 2007) Pep-1 KETWWETWWTEWSQPKKKRKV 21(Morris, Depollier et al., 2001) Pep-2 KETWFETWFTEWSQPKKKRKV 22(Morris, Chaloin et al., 2004) Xentry LCLRPVG 23(Montrose, Yang et al., 2013) Arginine stretch RRRRRRRRR 24(Zhou, Wu et al., 2009) Transportan WTLNSAGYLLGKINLKALAALAKKIL 25(Hallbrink, Floren et al., 2001) SynB1 RGGRLSYSRRRFSTSTGR 26(Drin, Cottin et al., 2003) SynB3 RRLSYSRRRF 27(Drin, Cottin et al., 2003) PTD4 YARAAARQARA 65 (Ho et al, 2001)

Without being bound by theory, cell-penetrating peptides are thought tointeract with the cell plasma membrane before crossing by pinocytosis orendocytosis. In the case of the TAT peptide, its hydrophilic nature andcharge are thought to promote its insertion within the plasma membraneand the formation of a pore (Herce and Garcia 2007). Alpha helix motifswithin hydrophobic peptides (such as SP) are also thought to form poreswithin plasma membranes (Veach, Liu et al., 2004).

In some embodiments, shuttle agents of the present description maycomprise one or more CPD or type of CPD. More particularly, they maycomprise at least 2, at least 3, at least 4, or at least 5 or more CPDs.In some embodiments, the shuttle agents can comprise between 1 and 10CPDs, between 1 and 6 CPDs, between 1 and 5 CPDs, between 1 and 4 CPDs,between 1 and 3 CPDs, etc.

In some embodiments, the CPD may be TAT having the amino acid sequenceof SEQ ID NO: 17, or a variant thereof having at least 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% identity to SEQ ID NO: 17and having cell penetrating activity; or Penetratin having the aminoacid sequence of SEQ ID NO: 18, or a variant thereof having at least70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, or 95% identity to SEQ ID NO: 18 and having cell penetratingactivity.

In some embodiments, the CPD may be PTD4 having the amino acid sequenceof SEQ ID NO: 65, or a variant thereof having at least 70%, 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81% 82%, 83%, 84%, 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% identity to SEQ ID NO:65.

In some embodiments, the order or placement of the CPD relative to theother domains (ELD, histidine-rich domains) within the shuttle agents ofthe present description may be varied provided the shuttling ability ofthe shuttle agent is retained.

In some embodiments, the CPD may be a variant or fragment of any onethose listed in Table II, and having cell penetrating activity. In someembodiments, the CPD may comprise or consist of the amino acid sequenceof any one of SEQ ID NOs: 16-27 or 65, or a sequence which is at least70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 85%, 90%, 91%, 92%, 93%, 94%, or 95%identical to any one of SEQ ID NOs: 16-27 or 65, and having cellpenetrating activity.

In some embodiments, shuttle agents of the present description do notcomprise any one of the amino acid sequences of SEQ ID NOs: 16-27 or 65.

Cargos

In some aspects, peptide shuttle agents of the present description maybe useful for delivering a polypeptide cargo (e.g., an independentpolypeptide cargo) from an extracellular space to the cytosol and/ornucleus of a target eukaryotic cell, wherein the synthetic peptideshuttle agent is used at a concentration sufficient to increase thetransduction efficiency of said polypeptide cargo, as compared to in theabsence of said synthetic peptide shuttle agent. In some embodiments,the polypeptide cargo may be fused to one or more CPDs to furtherfacilitate intracellular delivery. In some embodiments, the CPD fused tothe polypeptide cargo may be the same or different from a CPD that maybe present in the shuttle agent of the present description. Such fusionproteins may be constructed using standard recombinant technology. Insome embodiments, the independent polypeptide cargo may be fused,complexed with, or covalently bound to a second biologically activecargo (e.g., a biologically active polypeptide or compound).Alternatively or simultaneously, the polypeptide cargo may comprise asubcellular targeting domain.

In some embodiments, the polypeptide cargo must be delivered to thenucleus for it to carry out its intended biological effect One suchexample is when the cargo is a polypeptide intended for nuclear delivery(e.g., a transcription factor). In this regard, studies on themechanisms of translocation of viral DNA have led to the identificationof nuclear localization signals (NLSs). The NLS sequences are recognizedby proteins (importins α and β), which act as transporters and mediatorsof translocation across the nuclear envelope. NLSs are generallyenriched in charged amino acids such as arginine, histidine, and lysine,conferring a positive charge which is partially responsible for theirrecognition by importins. Accordingly, in some embodiments, thepolypeptide cargo may comprise an NLS for facilitating nuclear delivery,such as one or more of the NLSs as listed in Table III, or a variantthereof having nuclear targeting activity. Of course, it is understoodthat, in certain embodiments, the polypeptide cargo may comprise itsnatural NLS.

TABLE III Nuclear localizaion signals Name Amino acid sequenceSEQ ID NO: Reference(s) E1a KRPRP 28 (Kohler, Gorlich et al., 2001)SV40 T-Ag PKKKRKV 29 (Lanford, Kanda et al., 1986) c-myc PAAKRVKLD 30(Makkerh, Dingwall et al., 1996) Op-T-NLS SSDDEATADSQHAAPPKKKRKV 31(Chan and Jans 1999) Vp3 KKKRK 32 (Nakanishi, Shum et al., 2002)Nucleoplasmin KRPAATKKAGQAKKKK 33 (Fanara, Hodel et al., 2000)Histone 2B NLS DGKKRKRSRK 34 (Moreland, Langevin et al., 1987)Xenopus N1 VRKKRKTEEESPLKDKDAKKSKQE 35 (Kleinschmidt and Seiter 1988)PARP KRKGDEVDGVDECAKKSKK 36 (Schreiber, Molinete et al., 1992) PDX-1RRMKWKK 37 (Moede, Leibiger et al., 1999) QKI-5 RVHPYQR 38(Wu, Zhou et al., 1999) HCDA KRPACTLKPECVQQLLVCSQEAKK 39(Somasekaram, Jarmuz et al., 1999) H2B GKKRSKA 40(Moreland, Langevin et al., 1987) v-Rel KAKRQR 41(Gilmore and Temin 1988) Amida RKRRR 42 (Irie, Yamagata et al., 2000)RanBP3 PPVKRERTS 43 (Welch, Franke et al., 1999) Pho4p PYLNKRKGKP 44(Welch, Franke et al., 1999) LEF-1 KKKKRKREK 45(Prieve and Waterman 1999) TCF-1 KKKRRSREK 46 (Prieve and Waterman 1999)BDV-P PRPRKIPR 47 (Shoya, Kobayashi et al., 1998) TR2KDCVINKHHRNRCQYCRLQR 48 (Yu, Lee et al., 1998) SOX9 PRRRK 49(Sudbeck and Scherer 1997) Max PQSRKKLR 50 (Kato, Lee et al., 1992)

Once delivered to the cytoplasm, recombinant proteins are exposed toprotein trafficking system of eukaryotic cells. Indeed, all proteins aresynthesized in the cell's cytoplasm and are then redistributed to theirfinal subcellular localization by a system of transport based on smallamino acid sequences recognized by shuttle proteins (Karniely and Pines2005, Stojanovski, Bohnert et al., 2012). In addition to NLSs, otherlocalization sequences can mediate subcellular targeting to variousorganelles following intracellular delivery of the polypeptide cargos ofthe present description. Accordingly, in some embodiments, polypeptidecargos of the present description may comprise a subcellularlocalization signal for facilitating delivery of the shuttle agent andcargo to specific organelles, such as one or more of the sequences aslisted in Table IV, or a variant thereof having correspondingsubcellular targeting activity.

TABLE IV Subcellular localization signals Amino acid SEQ ID Namesequence NO: Reference(s) Mitochondrial signal sequence NLVERCFTD 51(Milenkovic, Ramming et al., 2009) from Tim9Mitochondrial signal sequence MLSLRQSIRFFK 52(Hurt, Pesold-Hurt et al., 1985) from Yeast cytochrome coxidase subunit IV Mitochondrial signal sequence MLISRCKWSRFPGNQR 53(Bejarano and Gonzalez 1999) from 18S rRNA Peroxisome signal sequence-SKL 54 (Gould, Keller et al., 1989) PTS1 Nucleolar signal sequenceMQRKPTIRRKNLRLRRK 55 (Scott, Boisvert et al., 2010) from BIRC5Nucleolar signal sequence KQAWKQKWRKK 56 (Scott, Boisvert et al., 2010)from RECQL4

In some embodiments, the cargo can be a biologically active compoundsuch as a biologically active (recombinant) polypeptide (e.g., atranscription factor, a cytokine, or a nuclease) intended forintracellular delivery. As used herein, the expression “biologicallyactive” refers to the ability of a compound to mediate a structural,regulatory, and/or biochemical function when introduced in a targetcell.

In some embodiments, the cargo may be a recombinant polypeptide intendedfor nuclear delivery, such as a transcription factor. In someembodiments, the transcription factor can be HOXB4 (Lu, Feng et al.,2007), NUP98-HOXA9 (Takeda, Goolsby et al., 2006), Oct3/4, Sox2, Sox9,Klf4, c-Myc (Takahashi and Yamanaka 2006), MyoD (Sung, Mun et al.,2013), Pdx1, Ngn3 and MafA (Akinci, Banga et al., 2012), Blimp-1 (Lin,Chou et al., 2013), Eomes, T-bet (Gordon, Chaix et al., 2012), FOXO3A(Warr, Binnewies et al., 2013), NF-YA (Dolfini, Minuzzo et al., 2012),SALL4 (Aguila, Liao et al., 2011), ISL1 (Fonoudi, Yeganeh et al., 2013),FoxA1 (Tan, Xie et al., 2010), Nanog, Esrrb, Lin28 (Buganim et al.,2014), HIF1-alpha (Lord-Dufour et al., 2009), Hlf, Runx1t1, Pbx1, Lmo2,Zfp37, Prdm5 (Riddell et al., 2014), or Bcl-6 (Ichii, Sakamoto et al.,2004).

In some embodiments, the cargo may be a recombinant polypeptide intendedfor nuclear delivery, such as a nuclease useful for genome editingtechnologies. In some embodiments, the nuclease may be an RNA-guidedendonuclease, a CRISPR endonuclease, a type I CRISPR endonuclease, atype II CRISPR endonuclease, a type III CRISPR endonuclease, a type IVCRISPR endonuclease, a type V CRISPR endonuclease, a type VI CRISPRendonuclease, CRISPR associated protein 9 (Cas9), Cpf1 (Zetsche et al.,2015), CasX and/or CasY (Burstein et al., 2016) a zinc-finger nuclease(ZFN), a Transcription activator-like effector nuclease (TALEN) (Cox etal., 2015), a homing endonuclease, a meganuclease, a DNA-guided nucleasesuch as Natronobacterium gregoryi Argonaute (NgAgo; Gao et al., 2016),or any combination thereof. In some embodiments, the nuclease may be acatalytically dead endonuclease, such as a catalytically dead CRISPRassociated protein 9 (dCas9), dCpf1, dCasX, dCasY, or any combinationthereof. Other nucleases not explicitly mentioned here may neverthelessbe encompassed in the present description. In some embodiments, thenuclease may be fused to a nuclear localization signal (e.g., Cas9-NLS;Cpf1-NLS; ZFN-NLS; TALEN-NLS). In some embodiments, the nuclease may becomplexed with a nucleic acid (e.g., one or more guide RNAs, a crRNA, atracrRNAs, or both a crRNA and a tracrRNA). In some embodiments, thenuclease may possess DNA or RNA-binding activity, but may lack theability to cleave DNA.

In some embodiments, the shuttle agents of the present description maybe used for intracellular delivery (e.g., nuclear delivery) of one ormore CRISPR endonucleases, for example one or more of the CRISPRendonucleases described below.

Type I and its subtypes A, B, C, D, E, F and I, including theirrespective Cas1, Cas2, Cas3, Cas4, Cas6, Cas7 and Cas8 proteins, and thesignature homologs and subunits of these Cas proteins including Cse1,Cse2, Cas7, Cas5, and Cas6e subunits in E. coli (type I-E) and Csy1,Csy2, Csy3, and Cas6f in Pseudomonas aeruginosa (type I-F) (Wiedenheftet al., 2011; Makarova et al, 2011). Type II and its subtypes A, B, C,including their respective Cas1, Cas2 and Cas9 proteins, and thesignature homologs and subunits of these Cas proteins including Csncomplexes (Makarova et al, 2011). Type III and its subtypes A, B andMTH326-like module, including their respective Cas1, Cas2, Cas6 andCas10 proteins, and the signature homologs and subunits of these Casproteins including Csm and CMR complexes (Makarova et al, 2011). Type IVrepresents the Csf3 family of Cas proteins. Members of this family showup near CRISPR repeats in Acidithiabacillus ferrooxidans ATCC 23270,Azoarcus sp. (strain EbN1), and Rhodoferax ferrireducens (strain DSM15236/ATCC BAA-621/T118). In the latter two species, the CRISPR/Caslocus is found on a plasmid. Type V and it subtypes have only recentlybeen discovered and include Cpf1, C2c1, and C2c3. Type VI includes theenzyme C2c2, which reported shares little homology to known sequences.

In some embodiments, the shuttle agents of the present description maybe used in conjunction with one or more of the nucleases, endonucleases,RNA-guided endonuclease, CRISPR endonuclease described above, for avariety of applications, such as those described herein. CRISPR systemsinteract with their respective nucleic acids, such as DNA binding, RNAbinding, helicase, and nuclease motifs (Makarova et al, 2011; Barrangou& Marraffini, 2014). CRISPR systems may be used for different genomeediting applications including:

-   -   a Cas-mediated genome editing method conducting to        non-homologous end-joining (NHEJ) and/or Homologous-directed        recombination (HDR) (Cong et al, 2013);    -   a catalytically dead Cas (dCas) that can repress and/or activate        transcription initiation when bound to promoter sequences, to        one or several gRNA(s) and to a RNA polymerase with or without a        complex formation with others protein partners (Bikard et al,        2013);    -   a catalytically dead Cas (dCas) that can also be fused to        different functional proteins domains as a method to bring        enzymatic activities at specific sites of the genome including        transcription repression, transcription activation, chromatin        remodeling, fluorescent reporter, histone modification,        recombinase system acetylation, methylation, ubiquitylation,        phosphorylation, sumoylation, ribosylation and citrullination        (Gilbert et al, 2013).

The person of ordinary skill in the art will understand that the presentshuttle agents, although exemplified with Cas9 and Cpf1 in the presentexamples, may be used with other nucleases as described herein. Thus,nucleases such as Cpf1, Cas9, and variants of such nucleases or others,are encompassed by the present description. It should be understoodthat, in one aspect, the present description may broadly cover any cargohaving nuclease activity, such an RNA-guided endonuclease, or variantsthereof (e.g., those that can bind to DNA or RNA, but have lost theirnuclease activity; or those that have been fused to a transcriptionfactor).

In some embodiments, the polypeptide cargo may be a cytokine such as achemokine, an interferon, an interleukin, a lymphokine, or a tumournecrosis factor. In some embodiments, the polypeptide cargo may be ahormone or growth factor. In some embodiments, the cargo may be anantibody (e.g., a labelled antibody, a therapeutic antibody, ananti-apoptotic antibody, an antibody that recognizes an intracellularantigen). In some embodiments, the cargo can be a detectable label(fluorescent polypeptide or reporter enzyme) that is intended forintracellular delivery, for example, for research and/or diagnosticpurposes.

In some embodiments, the cargo may be a globular protein or a fibrousprotein. In some embodiments, the cargo may have a molecule weight ofany one of about 5, 10, 15, 20, 25, 30, 35, 40, 45, to 50 to about 150,200, 250, 300, 350, 400, 450, 500 kDa or more. In some embodiments, thecargo may have a molecule weight of between about 20 to 200 kDa.

In some embodiments, the polypeptide cargo may be a peptide cargo, suchas peptide that recognizes an intracellular molecule.

In some embodiments, the polypeptide cargo may be an enzyme and/or anenzyme inhibitor.

In some embodiments, peptide shuttle agents of the present descriptionmay be useful for delivering a polypeptide cargo from an extracellularspace to the cytosol and/or nucleus of different types of targeteukaryotic cells, wherein the synthetic peptide shuttle agent is used ata concentration sufficient to increase the transduction efficiency ofsaid polypeptide cargo, as compared to in the absence of said syntheticpeptide shuttle agent. The target eukaryotic cells may be an animalcell, a mammalian cell, or a human cell. In some embodiments, the targeteukaryotic cells may be a stem cell (e.g., embryonic stem cells,pluripotent stem cells, induced pluripotent stem cells, neural stemcells, mesenchymal stem cells, hematopoietic stem cells, peripheralblood stem cells), a primary cell (e.g., myoblast, fibroblast), or animmune cell (e.g., NK cell, T cell, dendritic cell, antigen presentingcell). It will be understood that cells that are often resistant or notamenable to protein transduction may be interesting candidates for thesynthetic peptides or polypeptide-based shuttle agents of the presentdescription.

Non-Toxic, Metabolizable Shuttle Agents

In some embodiments, the shuttle agents of the present description maybe non-toxic to the intended target eukaryotic cells at concentrationsup to 50 μM, 45 μM, 40 μM, 35 μM, 30 μM, 25 μM, 20 μM, 15 μM, 10 μM, 9μM, 8 μM, 7 μM, 6 μM, 5 μM, 4 μM, 3 μM, 2 μM, 1 μM, 0.5 μMm 0.1 μM, or0.05 μM. Cellular toxicity of shuttle agents of the present descriptionmay be measured using any suitable method. Furthermore, transductionprotocols may be adapted (e.g., concentrations of shuttle and/or cargoused, shuttle/cargo exposure times, exposure in the presence or absenceof serum), to reduce or minimize toxicity of the shuttle agents, and/orto improve/maximize transfection efficiency.

In some embodiments, shuttle agents of the present description may bereadily metabolizable by intended target eukaryotic cells. For example,the shuttle agents may consist entirely or essentially of peptides orpolypeptides, for which the target eukaryotic cells possess the cellularmachinery to metabolize/degrade. Indeed, the intracellular half-life ofthe synthetic peptides and polypeptide-based shuttle agents of thepresent description is expected to be much lower than the half-life offoreign organic compounds such as fluorophores. However, fluorophorescan be toxic and must be investigated before they can be safely usedclinically (Alford et al., 2009). In some embodiments, shuttle agents ofthe present description may be suitable for clinical use. In someembodiments, the shuttle agents of the present description may avoid theuse of domains or compounds for which toxicity is uncertain or has notbeen ruled out.

Cocktails

In some embodiments, the present description relates to a compositioncomprising a cocktail of at least 2, at least 3, at least 4, or at least5 different types of the synthetic peptides or polypeptide-based shuttleagents as defined herein. In some embodiments, combining different typesof synthetic peptides or peptide shuttle agents (e.g., different shuttleagents comprising different types of domains) may provide increasedversatility for delivering different polypeptide cargos intracellularly.Furthermore, without being bound by theory, combining lowerconcentrations of different types of shuttle agents may help reducecellular toxicity associated with using a single type of shuttle agent(e.g., at higher concentrations).

Methods, Kits, Uses and Cells

In some embodiments, the present description relates to methods fordelivering a polypeptide cargo from an extracellular space to thecytosol and/or nucleus of a target eukaryotic cell. The methods comprisecontacting the target eukaryotic cell with the polypeptide cargo in thepresence of a shuttle agent at a concentration sufficient to increasethe transduction efficiency of said polypeptide cargo, as compared to inthe absence of said shuttle agent. In some embodiments, contacting thetarget eukaryotic cell with the polypeptide cargo in the presence of theshuttle agent results in an increase in the transduction efficiency ofsaid polypeptide cargo by at least 10-fold, 20-fold, 30-fold, 40-fold,50-fold, or 100-fold, as compared to in the absence of said shuttleagent.

In some embodiments, the present description relates to a method forincreasing the transduction efficiency of a polypeptide cargo to thecytosol of a target eukaryotic cell. As used herein, the expression“increasing transduction efficiency” refers to the ability of a shuttleagent of the present description to improve the percentage or proportionof a population of target cells into which a cargo of interest (e.g., apolypeptide cargo) is delivered intracellularly across the plasmamembrane. Immunofluorescence microscopy, flow cytometry, and othersuitable methods may be used to assess cargo transduction efficiency. Insome embodiments, a shuttle agent of the present description may enablea transduction efficiency of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, or 85%, for example as measure byimmunofluorescence microscopy, flow cytometry, FACS, and other suitablemethods. In some embodiments, a shuttle agent of the present descriptionmay enable one of the aforementioned transduction efficiencies togetherwish a cell viability of at least 25%, 30%, 35%, 40%, 45%, 50%, 55%,60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, for example as measure by theassay described in Example 3.3a, or by another suitable assay known inthe art.

In addition to increasing target cell transduction efficiency, shuttleagents of the present description may facilitate the delivery of a cargoof interest (e.g., a polypeptide cargo) to the cytosol of target cells.In this regard, efficiently delivering an extracellular cargo to thecytosol of a target cell using peptides can be challenging, as the cargooften becomes trapped in intracellular endosomes after crossing theplasma membrane, which may limit its intracellular availability and mayresult in its eventual metabolic degradation. For example, use of theprotein transduction domain from the HIV-1 Tat protein has been reportedto result in massive sequestration of the cargo into intracellularvesicles. In some aspects, shuttle agents of the present description mayfacilitate the ability of endosomally-trapped cargo to escape from theendosome and gain access to the cytoplasmic compartment. In this regard,the expression “to the cytosol” in the phrase “increasing thetransduction efficiency of an independent polypeptide cargo to thecytosol,” is intended to refer to the ability of shuttle agents of thepresent description to allow an intracellularly delivered cargo ofinterest to escape endosomal entrapment and gain access to thecytoplasmic compartment. After a cargo of interest has gained access tothe cytosol, it may be subsequently targeted to various subcellularcompartments (e.g., nucleus, nucleolus, mitochondria, peroxisome). Insome embodiments, the expression “to the cytosol” is thus intended toencompass not only cytosolic delivery, but also delivery to othersubcellular compartments that first require the cargo to gain access tothe cytoplasmic compartment.

In some embodiments, the methods of the present description are in vitromethods. In other embodiments, the methods of the present descriptionare in vive methods.

In some embodiments, the methods of the present description may comprisecontacting the target eukaryotic cell with the shuttle agent, orcomposition as defined herein, and the polypeptide cargo. In someembodiments, the shuttle agent, or composition may be pre-incubated withthe polypeptide cargo to form a mixture, prior to exposing the targeteukaryotic cell to that mixture. In some embodiments, the type ofshuttle agent may be selected based on the amino acid sequence of thepolypeptide cargo to be delivered intracellularly. In other embodiments,the type of shuttle agent may be selected to take into account the aminoacid sequence of the polypeptide cargo to be delivered intracellularly,the type of cell, the type of tissue, etc.

In some embodiments, the method may comprise multiple treatments of thetarget cells with the shuttle agent, or composition (e.g., 1, 2, 3, 4 ormore times per day, and/or on a pre-determined schedule). In such cases,lower concentrations of the shuttle agent, or composition may beadvisable (e.g., for reduced toxicity). In some embodiments, the cellsmay be suspension cells or adherent cells. In some embodiments, theperson of skill in the art will be able to adapt the teachings of thepresent description using different combinations of shuttles, domains,uses and methods to suit particular needs of delivering a polypeptidecargo to particular cells with a desired viability.

In some embodiments, the methods of the present description may apply tomethods of delivering a polypeptide cargo intracellularly to a cell invivo. Such methods may be accomplished by parenteral administration ordirect injection into a tissue, organ, or system.

In some embodiments, the shuttle agent, or composition, and thepolypeptide cargo may be exposed to the target cell in the presence orabsence of serum. In some embodiments, the method may be suitable forclinical or therapeutic use.

In some embodiments, the present description relates to a kit fordelivering a polypeptide cargo from an extracellular space to thecytosol and/or nucleus of a target eukaryotic cell. In some embodiments,the present description relates to a kit for increasing the transductionefficiency of a polypeptide cargo to the cytosol of a target eukaryoticcell. The kit may comprise the shuttle agent, or composition as definedherein, and a suitable container.

In some embodiments, the target eukaryotic cells may be an animal cell,a mammalian cell, or a human cell. In some embodiments, the targeteukaryotic cells may be a stem cell (e.g., embryonic stem cells,pluripotent stem cells, induced pluripotent stem cells, neural stemcells, mesenchymal stem cells, hematopoietic stem cells, peripheralblood stem cells), a primary cell (e.g., myoblast, fibroblast), or animmune cell (e.g., NK cell, T cell, dendritic cell, antigen presentingcell). In some embodiments, the present description relates to anisolated cell comprising a synthetic peptide or polypeptide-basedshuttle agent as defined herein. In some embodiments, the cell may be aprotein-induced pluripotent stem cell. It will be understood that cellsthat are often resistant or not amenable to protein transduction may beinteresting candidates for the synthetic peptides or polypeptide-basedshuttle agents of the present description.

In some embodiments, the present description relates to a method forproducing a synthetic peptide shuttle agent that delivers a polypeptidecargo from an extracellular space to the cytosol and/or nucleus of atarget eukaryotic cell, the method comprising synthesizing a peptidewhich is:

-   -   (1) a peptide at least 20 amino adds in length comprising    -   (2) an amphipathic alpha-helical motif having    -   (3) a positively-charged hydrophilic outer face, and a        hydrophobic outer face,        wherein at least five of the following parameters (4) to (15)        are respected:    -   (4) the hydrophobic outer face comprises a highly hydrophobic        core consisting of spatially adjacent L, I, F, V, W, and/or M        amino acids representing 12 to 50% of the amino acids of the        peptide, based on an open cylindrical representation of the        alpha-helix having 3.6 residues per turn;    -   (5) the peptide has a hydrophobic moment (μ) of 3.5 to 11;    -   (6) the peptide has a predicted net charge of at least +4 at        physiological pH;    -   (7) the peptide has an isoelectric point (pI) of 8 to 13;    -   (8) the peptide is composed of 35% to 65% of any combination of        the amino acids: A, C, G, I, L, M, F, P, W, Y, and V;    -   (9) the peptide is composed of 0% to 30% of any combination of        the amino acids: N, Q, S, and T;    -   (10) the peptide is composed of 35% to 85% of any combination of        the amino acids A, L, K, or R;    -   (11) the peptide is composed of 15% to 45% of any combination of        the amino acids: A and L, provided there being at least 5% of L        in the peptide;    -   (12) the peptide is composed of 20% to 45% of any combination of        the amino acids K and R;    -   (13) the peptide is composed of 0% to 10% of any combination of        the amino acids: D and E;    -   (14) the difference between the percentage of A and L residues        in the peptide (% A+L), and the percentage of K and R residues        in the peptide (% K+R), is less than or equal to 10%; and    -   (15) the peptide is composed of 10% to 45% of any combination of        the amino adds: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and        H.

In some embodiments, the present description relates to a method foridentifying a shuttle agent that delivers a polypeptide cargo from anextracellular space to the cytosol and/or nucleus of a target eukaryoticcell, the method comprising: (a) synthesizing a peptide which is thepeptide as defined herein; (b) contacting the target eukaryotic cellwith the polypeptide cargo in the presence of said peptide; (c)measuring the transduction efficiency of the polypeptide cargo in thetarget eukaryotic cell; and (d) identifying the peptide as being ashuttle agent that transduces the polypeptide cargo, when an increase inthe transduction efficiency of said polypeptide cargo in the targeteukaryotic cell is observed.

In some embodiments, the present description relates to a genome editingsystem comprising: (a) the shuttle agent as defined herein; (b) aCRISPR-associated endonuclease; and (c) one or more guide RNAs. In someembodiments, the genome editing system may further comprise a linear DNAtemplate for controlling the genome editing.

Genome Editing for Improved Cell Therapy

In some embodiments, the shuttle agents, synthetic peptides,compositions, and methods described herein may be used for transducinggenome-editing complexes (e.g., the CRISPR-based genome editingcomplexes) to genetically engineer cells for improved cell therapy, ascompared to native cells or unengineered cells. Such improvements mayinclude, for example, reducing the immunogenicity of the engineeredcells and/or improving the activity/efficacy of the engineered cells.

Particularly attractive immune cells for genome engineering may benatural killer (NK) cells, given their natural ability to recognize andkill tumor cells. Accordingly, in some embodiments, the presentdescription relates to the use of the shuttle agents, syntheticpeptides, compositions, and methods described herein for transducinggenome-editing complexes (e.g., the CRISPR-based genome editingcomplexes) to genetically engineer NK (or other immune cells that wouldbenefit from the same modifications) for improved cell-basedimmunotherapy. For example, the present description may relate to theintracellular delivery of one or more CRISPR-based genome editingcomplexes that comprise a guide RNA and/or linear DNA template targetingthe CBLB gene, c-CBL gene, GSK3 gene, ILT2 gene, CISH gene, NKG2a gene,B2M gene, or any combination thereof. Such gene targets may potentiateNK-mediated cellular cytotoxicity following knockout, as discussedbelow.

1. NKG2A (KLRC1, CD159A, Killer Cell Lectin-Like Receptor C1)

CD94/NKG2A acts as an MHC class-I specific NK inhibitory receptor (Braudet al., 1998; Lee et al., 1998). It is expressed by a subset of NK cellsknown as CD56^(bright) CD16^(dim) (˜10% of peripheral NK), which aretypically less cytotoxic (Cooper et al., 2001; Poli et al., 2009). NKG2Aligands are the non-classical MHC class-I HLA-E molecules that areexpressed in every human cell. The recognition of HLA-E by the NKG2Areceptor is part of the “self-tolerance” mechanism (also including KIRreceptors), resulting in negative modulation of NK cell cytotoxicity(Lee et al., 1998).

There exists clinical evidence demonstrating the role of non-classicalHLA class I, mainly HLA-E and HLA-G (see ILT-2 target), in evadingimmune surveillance resulting in higher cancer relapses and decreaseoverall survival following surgery (de Kruijf et al., 2010; Levy et al.,2008; Ye et al., 2007a; Yie et al., 2007b; Yie et al., 2007c; Yie etal., 2007d; Guo et al., 2015; Ishigami et al., 2015; Zhen et al., 2013).The use of NKG2A-KO NK cells during adoptive cell therapy may counteractthe presence of HLA-E molecules (membrane-bound or solubles) in tumormicroenvironment. In addition, NK cells expanded from IL15 orIL21-expressing K562 feeder cells lead to a high percentage of NKG2AJcells (Denman et al., 2012), and it may be desirable to knockout thisinhibitory receptor during the expansion process. Furthermore, theresults in Example G.9 demonstrate that NKG2A-KO NK92 cells aresignificantly more cytotoxic against IFN-gamma-treated HeLa cells.

2. ILT2 (Ig-Like Transcript 2 Gene)

ILT2 is an inhibitory receptor expressed on several immune cells,including NK cells (Kirwan et al., 2005). The ligands for this receptorare HLA-G molecules, which are naturally expressed only in thymus andtrophoblasts. However, many tumors gain the capacity to express HLA-G toescape immune cell attack by inhibition through ILT2 receptoractivation. In fact, NKL^(ILT2−) cells are more potent than parental NKLagainst HLA-G-overexpressing K562 cells (Wu et al., 2015). Moreover,overexpression of HLA-G in OVCAR-3 cancer cells impaired NKcell-mediated cytotoxicity (Lin et al., 2007). As for HLA-E, expressionof HLA-G on cancer cells is generally associated with poor prognosis.

3. c-Cbl and Cbl-b (Casitas B-Lineage Lymphoma Proto-Oncogene Family).

These genes (from the Casitas B-lineage lymphoma proto-oncogene, Cblfamily) encode for E3 ligases, which are function in the proteinubiquitylation pathway (regulation of cellular protein content). E3ligases catalyze the formation of a covalent bond between Ub (ubiquitin)and specific lysine residues on targeted proteins (more than thousand E3ligases in mammalians). Cbl family members are involved in negativeregulation of signaling by receptor tyrosine kinases on immune cells bybinding and ubiquitylating phosphorylated receptor and adaptors (Liu etal., 2014; utz-Nicoladoni, 2015). One demonstrated that both c-cbl andCbl-b ubiquitylate phosphorylated LAT adaptor. Phosphorylation of LATfollowing NK cell activation is required to recruit other mediators,especially PLC-□□□ and siRNA-mediated c-cbl and Cbl-b knockdownincreased NK cell activity against B cell lymphoma 721.221-Cw4 (Matalonet al., 2016).

Others identified TAM (Tyro3, Axl, Mer) receptors as targets for Cbl-bubiquitylation (Paolino et al., 2014). However, assuming that TAMreceptors are proposed to negatively regulate NK cells, Cbl-b knockoutshould rather be associated to a decrease in NK cell activity.Therefore, TAM receptors may be considered as a good target to enhanceNK cells but unlikely via Cbl-b knockout.

In vivo studies demonstrated that Cbl-b^(−/−) mice prevent primary tumorgrowth (Loeser et al., 2007). In addition, NK cells isolated from thesemice have increased proliferation and IFN-□□ production when activated(Paolino et al., 2014).

4. GSK3B (Glycogen Synthase Kinase Beta)

GSK3b is a Ser/Thr kinase involved in several cellular functions, suchas proliferation, apoptosis, inflammatory response, stress, and others(Patel et al., 2017). Inhibition of GSK3b (using small inhibitors) in NKcells leads to increase cytotoxicity (likely through IFN-g, TNF-□production, 2B4 stimulation and up-regulation of LFA-1) against AML(OCI-AML3) (Parameswaran et al., 2016; Aoukaty et al., 2005). We haverecently demonstrated that the GSK30 inhibitor, SB216763, enhances thecytotoxic activity of NK92 against HeLa cells (data not shown). Thiseffect is increased by co-incubation with IL-15.

5. CISH (Cytokine-Inducible SH2-Containing Protein)

CIS protein is a member of the suppressor of cytokine signaling (SOCS)proteins, which bind to phosphorylated JAKs and inhibit JAK-STATsignaling pathways. Recently, Cish−/− mice demonstrated that CIS is akey suppressor of IL15 signaling in NK cells (Delconte et al., 2016).Following IL15 exposure, these cells have prolonged IL15 responses, anelevated IFN-g production, and an increased cytotoxic potential.Moreover, there is a clear relationship between IL15 responsiveness andNKG2D-dependent cytotoxicity (Homg et al., 2007).

In clinical trials, co-injection of cytokines, such as IL2 and IL15,during adoptive NK-cell therapy is strongly recommended to sustain NKcell activity. However, such a co-injection induces serious side effectsto patients. The use of IL15-hypersensitive NK cells (CISH knockout)would benefit the treatment.

In some embodiments, disrupting the B2M gene encoding β2 microglobulin(B2M), a component of MHC class I molecules, may substantially reducethe immunogenicity of every cell expressing MHC class I. In otheraspects, the genome of NK cells can be modified after the delivery of agenome editing system as described herein. More specifically, thecytotoxicity of NK cells can be improved after the delivery of a genomeediting system targeting specific putative targets that may potentiateNK-mediated cellular cytotoxicity such as the NKG2A, ILT2, c-Cbl, Cbl-b,GSK3B and CISH genes.

Co-Transduction with a Polypeptide Cargo of Interest and a MarkerProtein

The ability of domain-based and rationally-designed peptide shuttleagents to co-transduce two different polypeptide cargos into apopulation of target eukaryotic cells has been demonstrated herein andin PCT patent application publication No. WO/2016/161516. Example I ofthe present description shows that co-transducing a polypeptide cargo ofinterest (e.g., a CRISPR-endonuclease) and an independent marker protein(e.g., GFP) in a population of target eukaryotic cells may notnecessarily increase the overall transduction efficiency of thepolypeptide cargo of interest. However, it was surprisingly discoveredthat a strikingly high proportion of target eukaryotic cells that weresuccessfully transduced with the polypeptide cargo of interest, werealso successfully transduced with the marker protein. Conversely, astrikingly high proportion of cells that were not transduced with thepolypeptide cargo of interest, were also not transduced with the markerprotein. Isolating cells positive for the marker protein (e.g., viaFACS) resulted in a significant increase in the proportion of cells thatwere successfully transduced with the polypeptide cargo of interest, andthe correlation was found to be concentration dependent in that cellpopulations exhibiting the highest fluorescence of the marker proteinalso exhibited the highest proportion of transduction with thepolypeptide cargo of interest.

In some aspects, the present description relates to a method forenriching eukaryotic cells transduced with a polypeptide cargo ofinterest. The method may comprise (a) co-transducing a target eukaryoticcell population with a polypeptide cargo of interest and a markerprotein; and (b) isolating or concentrating eukaryotic cells transducedwith the marker protein, thereby enriching eukaryotic cells transducedwith the polypeptide cargo of interest.

In some embodiments, the marker protein may not be covalently bound tothe polypeptide cargo of interest (e.g., the marker protein isindependent from the polypeptide cargo of interest), the marker proteinmay be covalently bound to the polypeptide cargo of interest, the markerprotein may be non-covalently bound to the polypeptide cargo of interest(e.g., electrostatically and/or conformationally bound via a proteindomain-protein domain interaction), or the marker protein is covalentlybound to the polypeptide cargo of interest via a cleavable linker (e.g.,a linker peptide comprising an enzyme cleavage site, such as an enzymeexpressed in endosomes, lysosomes, or in the cytosol). In someembodiments, the intracellular concentration of the transduced markerprotein may be positively correlated with the intracellularconcentration of the transduced polypeptide cargo of interest.

In some embodiments, the marker protein may comprise a detectable label.As used herein, “detectable label” refers to a molecule or particle thatenables a person of skill in the art to identify and separate cellscomprising the label from cells lacking the label. In some embodiments,the marker protein may be a fluorescent protein, a fluorescently-labeledprotein, a bioluminescent protein, an isotopically-labelled protein, amagnetically-labeled protein, or another detectable label that enablesseparation of cells containing the marker protein from cells lacking themarker protein, or enables separation of cells based on the level ofintracellular marker protein.

In some embodiments, eukaryotic cells transduced with the marker proteinmay be isolated or concentrated using flow cytometry,fluorescence-activated cell sorting (FACS), magnetic-activated cellsorting (MACS), or other known cell separation/sorting technologies.Isolating or enriching successfully transduced cells may be particularlyadvantageous, for example, for polypeptide cargoes such as functionalCRISPR-based genome-editing complexes, which may be associated withrelatively lower transduction efficiencies.

It was also surprisingly disclosed herein in Example I that cells thatwere unsuccessfully transduced following a first round of transductionwith a polypeptide cargo of interest, may be isolated and re-transducedwith the polypeptide cargo of interest in subsequent rounds oftransduction. These results suggest that, although the cellsunsuccessfully transduced following a first round of transduction with apolypeptide cargo of interest are not necessarily refractory tosubsequent transductions.

In some embodiments, the present description relates to a method forenriching eukaryotic cells transduced with a polypeptide cargo ofinterest, the method comprising: (a) co-transducing a target eukaryoticcell population with a polypeptide cargo of interest and a markerprotein; (b) isolating eukaryotic cells transduced with the markerprotein from cells lacking the marker protein, thereby producing amarker protein-positive cell population and a marker protein-negativecell population. In some embodiments, steps (a) and (b) may be repeated,one or more times, on the marker protein-negative cell population, onthe marker protein-positive cell population, or on both the markerprotein-negative and the marker protein-positive cell populations. Insome embodiments, the method for enriching eukaryotic cells transducedwith a polypeptide cargo of interest may be automated, for example, byisolating cells negative for transduction with the marker protein andre-transducing the marker protein-negative cell population.

In some embodiments, the present description relates to a method forenriching eukaryotic cells transduced with a polypeptide cargo ofinterest, the method comprising: (a) co-transducing a target eukaryoticcell population with a polypeptide cargo of interest and a markerprotein; and (b) isolating eukaryotic cells transduced with the markerprotein based on their intracellular concentration of the markerprotein.

In some embodiments, the marker protein described herein may be aprotein that stimulates cell proliferation (e.g., a growth factor or atranscription factor), a protein that stimulates cell differentiation, aprotein that promotes cell survival, an anti-apoptotic protein, or aprotein having another biological activity.

In some embodiments, the polypeptide cargo of interest and the markerprotein may be co-transduced by contacting the target eukaryotic cellwith the polypeptide cargo and the marker protein in the presence of apeptide transduction agent, wherein the peptide transduction agent ispresent at a concentration sufficient to increase the transductionefficiency of the polypeptide cargo and the marker protein, as comparedto in the absence of said peptide transduction agent. In someembodiments, the peptide transduction agent may be an endosomolyticpeptide. In some embodiments, the peptide transduction agent is orcomprises a domain-based peptide shuttle agent, or a rationally designedpeptide shuttle agent as defined herein, in PCT patent applicationpublication No. WO/2016/161516, and/or U.S. Pat. No. 9,738,687. In someembodiments, the aforementioned domain-based peptide shuttle agent maybe a synthetic peptide comprising an endosome leakage domain (ELD)operably linked to a cell penetrating domain (CPD), or an ELD operablylinked to a histidine-rich domain and a CPD. In some embodiment, theaforementioned domain-based peptide shuttle agent: (a) comprises aminimum length of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 aminoacid residues and a maximum length of 35, 40, 45, 50, 55, 65, 70, 75,80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150amino acid residues; (b) has a predicted net charge of at least +4, +5,+6, +7, +8, +9, +10, +11, +12, +13, +14, or +15 at physiological pH; (c)is soluble in aqueous solution; or (d) any combination of (a) to (c). Insome embodiments, the ELD, CPD, histidine-rich domain, linker domain,are as defined herein. In some embodiments, the target eukaryotic cellscomprise or consist of animal cells, mammalian cells, human cells, stemcells, primary cells, immune cells, T cells, NK cells, dendritic cells,or other types or sub-types of cells.

In some embodiments, the polypeptide cargo of interest may be: (i) thepolypeptide cargo as defined herein; and/or (ii) one or moreCRISPR-associated endonucleases alone or with one or more correspondingguide RNA and/or linear DNA templates as defined herein.

Items

In some embodiments, the present description may relate to the followingitems:

1. A method for delivering a polypeptide cargo from an extracellularspace to the cytosol and/or nucleus of a target eukaryotic cell, saidmethod comprising contacting the target eukaryotic cell with thepolypeptide cargo in the presence of a shuttle agent at a concentrationsufficient to increase the transduction efficiency of said polypeptidecargo, as compared to in the absence of said shuttle agent, wherein saidshuttle agent is

-   -   (1) a peptide at least 20 amino acids in length comprising    -   (2) an amphipathic alpha-helical motif having    -   (3) a positively-charged hydrophilic outer face, and a        hydrophobic outer face,        wherein at least five of the following parameters (4) to (15)        are respected:    -   (4) the hydrophobic outer face comprises a highly hydrophobic        core consisting of spatially adjacent L, I, F, V, W, and/or M        amino acids representing 12 to 50% of the amino acids of the        peptide, based on an open cylindrical representation of the        alpha-helix having 3.6 residues per turn;    -   (5) the peptide has a hydrophobic moment (μ) of 3.5 to 11;    -   (6) the peptide has a predicted net charge of at least +4 at        physiological pH;    -   (7) the peptide has an isoelectric point (pI) of 8 to 13;    -   (8) the peptide is composed of 35% to 65% of any combination of        the amino acids: A, C, G, I, L, M, F, P, W, Y, and V;    -   (9) the peptide is composed of 0% to 30% of any combination of        the amino acids: N, Q, S, and T;    -   (10) the peptide is composed of 35% to 85% of any combination of        the amino acids: A, L, K, or R;    -   (11) the peptide is composed of 15% to 45% of any combination of        the amino acids: A and L, provided there being at least 5% of L        in the peptide;    -   (12) the peptide is composed of 20% to 45% of any combination of        the amino acids: K and R;    -   (13) the peptide is composed of 0% to 10% of any combination of        the amino acids: D and E;    -   (14) the difference between the percentage of A and L residues        in the peptide (% A+L), and the percentage of K and R residues        in the peptide (K+R), is less than or equal to 10%; and    -   (15) the peptide is composed of 10% to 45% of any combination of        the amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T and        H.        2. The method of item 1, wherein the shuttle agent respects at        least six, at least seven, at least eight, at least nine, at        least ten, at least eleven, or respects all of parameters (4) to        (15).        3. The method of item 1 or 2, wherein:    -   (i) said shuttle agent is a peptide having a minimum length of        20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, and a        maximum length of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,        51, 52, 53, 54, 55, 60, 65, 70, 80, 90, 100, 110, 120, 130, 140,        or 150 amino acids;    -   (ii) said amphipathic alpha-helical motif has a hydrophobic        moment (μ) between a lower limit of 3.5, 3.6, 3.7, 3.8, 3.9,        4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2,        5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5,        6.6, 6.7, 6.8, 6.9, 7.0, and an upper limit of 9.5, 9.6, 9.7,        9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5, 10.6, 10.7, 10.8,        10.9, or 11.0;    -   (iii) said amphipathic alpha-helical motif comprises a        positively-charged hydrophilic outer face comprising: (a) at        least two, three, or four adjacent positively-charged K and/or R        residues upon helical wheel projection; and/or (b) a segment of        six adjacent residues comprising three to five K and/or R        residues upon helical wheel projection, based on an alpha helix        having angle of rotation between consecutive amino acids of 100        degrees and/or an alpha-helix having 3.6 residues per turn;    -   (iv) said amphipathic alpha-helical motif comprises a        hydrophobic outer face comprising: (a) at least two adjacent L        residues upon helical wheel projection; and/or (b) a segment of        ten adjacent residues comprising at least five hydrophobic        residues selected from: L, I, F, V, W, and M, upon helical wheel        projection, based on an alpha helix having angle of rotation        between consecutive amino acids of 100 degrees and/or an        alpha-helix having 3.6 residues per turn;    -   (v) said hydrophobic outer face comprises a highly hydrophobic        core consisting of spatially adjacent L, I, F, V, W, and/or M        amino acids representing from 12.5%, 13%, 13.5%, 14%, 14.5%,        15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%, 19%, 19.5%, or        20%, to 25%, 30%, 35%, 40%, or 45% of the amino acids of the        peptide;    -   (vi) said peptide has a hydrophobic moment (μ) between a lower        limit of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,        5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3,        6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, and an upper limit of 9.5,        9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, or 10.5;    -   (vii) said peptide has a predicted net charge of between +4,        0.6, +6, +7, +8, +9, to +10, +11, +12, +13, +14, or +15;    -   (viii) said peptide has a predicted pI of 10-13; or    -   (ix) any combination of (i) to (viii).        4. The method of any one of items 1 to 3, wherein said shuttle        agent respects at least one, at least two, at least three, at        least four, at least five, at least six, or all of the following        parameters:    -   (8) the peptide is composed of 36% to 64%, 37% to 63%, 38% to        62%, 39% to 61%, or 40% to 60% of any combination of the amino        acids: A, C, G, I, L, M, F, P, W, Y, and V;    -   (9) the peptide is composed of 1% to 29%, 2% to 28%, 3% to 27%,        4% to 26%, 5% to 25%, 6% to 24%, 7% to 23%, 8% to 22%, 9% to        21%, or 10% to 20% of any combination of the amino acids: N, Q,        S, and T;    -   (10) the peptide is composed of 36% to 80%, 37% to 75%, 38% to        70%, 39% to 65%, or 40% to 60% of any combination of the amino        acids A, L, K, or R;    -   (11) the peptide is composed of 15% to 40%, 20% to 40%, 20 to        35%, or 20 to 30% of any combination of the amino acids: A and        L;    -   (12) the peptide is composed of 20% to 40%, 20 to 35%, or 20 to        30% of any combination of the amino acids: K and R;    -   (13) the peptide is composed of 5 to 10% of any combination of        the amino acids: D and E;    -   (14) the difference between the percentage of A and L residues        in the peptide (% A+L), and the percentage of K and R residues        in the peptide (K+R), is less than or equal to 9%, 8%, 7%, 6% or        5%; and    -   (15) the peptide is composed of 15 to 40%, 20% to 35%, or 20% to        30% of any combination of the amino acids: Q, Y, W, P, I, S, G,        V, F, E, D, C, M, N, T, and H.        5. The method of any one of items 1 to 4, wherein said peptide        comprises a histidine-rich domain.        6. The method of item 5, wherein said histidine-rich domain is:    -   (i) positioned towards the N terminus and/or towards the C        terminus of the peptide;    -   (ii) is a stretch of at least 3, at least 4, at least 5, or at        least 6 amino acids comprising at least 50%, at least 55%, at        least 60%, at least 65%, at least 70%, at least 75%, at least        80%, at least 85%, or at least 90% histidine residues; and/or        comprises at least 2, at least 3, at least 4, at least 5, at        least 6, at least 7, at least 8, or at least 9 consecutive        histidine residues; or    -   (iii) both (i) and (ii).        7. The method of any one of items 1 to 6, wherein said peptide        comprises a flexible linker domain rich in serine and/or glycine        residues.        8. The method of any one of items 1 to 7, wherein said peptide        comprises or consists of the amino acid sequence of: (a)        [X1]−[X2]−[linker]−[X3]−[X4] (Formula 1); (b)        [X1]−[X2]−[linker]−[X4]−[X3] (Formula 2); (c)        [X2]−[X1]−[linker]−[X3]−[X4] (Formula 3); (d)        [X2]−[X1]−[linker]−[X4]−[X3] (Formula 4); (e)        [X3]−[X4]−[linker]−[X1]−[X2] (Formula 5); (f)        [X3]−[X4]−[linker]−[X2]−[X1] (Formula 6); (g)        [X4]−[X3]−[linker]−[X1]−[X2] (Formula 7); or (h)        [X4]−[X3]−[linker]−[X2]−[X1] (Formula 8), wherein: [X1] is        selected from: 2[ϕ]−1[+]−2[ϕ]−1[ζ]−1[+]−; 2[ϕ]−1[+]−2[ϕ]−2[+]−;        1[+]−1[ϕ]−1[+]−2[ϕ]−1[ζ]−1[+]−; and 1[+]−1[ϕ]−1[+]−2[ϕ]−2[+]−;        [X2] is selected from: −2[ϕ]−1[+]−2[ϕ]−2−[ζ]−;        −2[ϕ]−1[+]−2[ϕ]−2[+]−; −2[ϕ]−1[+]−2[ϕ]−1[+]−1[ζ]−;        −2[ϕ]−1[+]−2[ϕ]−1[ζ]−1[+]−; −2[ϕ]−2[+]−1[ϕ]−2[+]−;        −2[ϕ]−2[+]−1[ϕ]−2[ζ]−; −2[ϕ]−2[+]−1[ϕ]−1[+]−1[ζ]−; and        −2[ϕ]−2[+]−1[ϕ]−1[ζ]−1[+]−; [X3] is selected from: −4[+]−A−;        −3[+]−G−A−; −3[+]−A−A−; −2[+]−1[ϕ]−1[+]−A−; −2[+]−1[ϕ]−G−A−;        −2[+]−1[ϕ]−A−A−; or −2[+]−A−1[+]−A; −2[+]−A−G−A; −2[+]−A−A−A−;        −1[ϕ]−3[+]−A−; −1[ϕ]−2[+]−G−A−; −1[ϕ]−2[+]−A−A−;        −1[ϕ]−1[+]−1[ϕ]−1[+]−A; −1[ϕ]−1[+]−1[ϕ]−G−A;        −1[ϕ]−1[+]−1[ϕ]−A−A−; 1[ϕ]−1[+]−A−1[+]−A; −1[ϕ]−1[+]−A−G−A;        −1[ϕ]−1[+]−A−A−A; −A−1[+]−A−1[+]−A; −A−1[+]−A−G−A; and        −A−1[+]−A−A−A; [X4] is selected from: −1[ζ]−2A−1[+]−A;        −1[ζ]−2A−2[+]; −1[+]−2A−1[+]−A; −1[ζ]−2A−1[+]−1[ζ]−A−1[+];        −1[ζ]−A−1[ϕ]−A−1[+]; −2[+]−A−2[+]; −2[+]−A−1[+]−A;        −2[+]−A−1[+]−−1[ζ]−A−1[+]; −2[+]−1[ζ]−A−1[+];        −1[+]−1[ζ]−A−1[+]−A; −1[+]−1[ζ]−A−2[+];        −1[+]−1[ζ]−A−1[+]−1[ζ]−A−1[+]; −1[+]−2[ζ]−A−1[+];        −1[+]−2[ζ]−2[+]; −1[+]−2[ζ]−1[+]−A; −1[+]−2[ζ]−1[+]−1[ζ]−A−1[+];        −1[+]−2[ζ]−1[ζ]−A−1[+]; −3[ζ]−2[+]; −3[ζ]−1[+]−A;        −3[ζ]−1[+]−1[ζ]−A−1[+]; −1[ζ]−2A−1[+]−A; −1[ζ]−2A−2[+];        −1[ζ]−2A−1[+]−1[ζ]−A−1[+]; −2[+]−A−1[+]−A; −2[+]−1[ζ]−1[+]−A;        −1[+]−1[ζ]−A−1[+]−A; −1[+]−2A−1[+]−1[ζ]−A−1[+]; and        −1[ζ]−A−1[ζ]−A−1[+]; and [linker] is selected from: -Gn-; −Sn-;        −(GnSn)n-; −(GnSn)nGn-; −(GnSn)nSn-; −(GnSn)nGn(GnSn)n-; and        -(GnSn)nSn(GnSn)n-; wherein: [ϕ] is an amino acid which is: Leu,        Phe, Trp, lie, Met, Tyr, or Val; [+] is an amino add which is:        Lys or Arg; [ζ] is an amino acid which is: Gln, Asn, Thr, or        Ser; A is the amino add Ala; G is the amino acid Gly; S is the        amino acid Ser and n is an integer from 1 to 20, 1 to 19, 1 to        18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to        11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 1 to        4, or 1 to 3.        9. The method of any one of items 1 to 8, wherein:    -   (i) said peptide is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%,        85%, 90%, or 95% identical to the amino acid sequence of any one        of SEQ ID NOs: 104, 105, 107, 108, 110-131, 133-135, 138, 140,        142, 145, 148, 151, 152, 169-242, and 243-10 242; or said        peptide comprises or consists of a functional variant of any one        of SEQ ID NOs: 104, 105, 107, 108, 110-131, 133-135, 138, 140,        142, 145, 148, 151, 152, 169-242, and 243-10 242;    -   (ii) said peptide:        -   (a) comprises or consists of the amino acid sequence of any            one of SEQ ID NOs: 104, 105, 107, 108, 110-131, 133-135,            138, 140, 142, 145, 148, 151, 152, 169-242, and 243-10 242;        -   (b) comprises the amino acid sequence motifs of SEQ ID NOs:            158 and/or 159; or        -   (c) comprises the amino acid sequence motif of SEQ ID NO:            158 operably linked to the amino acid sequence motif of SEQ            ID NO: 159; or    -   (iii) both (i) and (ii).        10. The method of any one of items 1 to 9, wherein the peptide        comprises an endosome leakage domain (ELD), and/or a cell        penetrating domain (CPD).        11. The method of item 10, wherein:    -   (i) said ELD is or is from: an endosomolytic peptide; an        antimicrobial peptide (AMP); a linear cationic alpha-helical        antimicrobial peptide; a Cecropin-A/Melittin hybrid (CM series)        peptide; pH-dependent membrane active peptide (PAMP); a peptide        amphiphile; a peptide derived from the N terminus of the HA2        subunit of influenza hemagglutinin (HA); CM18; Diphtheria toxin        T domain (DT); GALA; PEA; INF-7; LAH4; HGP; H5WYG; HA2; EB1;        VSVG; Pseudomonas toxin; melittin; KALA; JST-1; C(LLKK)₃C;        G(LLKK)₃G; or any combination thereof (ii) said CPD is or is        from: a cell-penetrating peptide or the protein transduction        domain from a cell-penetrating peptide; TAT; PTD4; Penetratin        (Antennapedia); pVEC; M918; Pep-1; Pep-2; Xentry; arginine        stretch; transportan; SynB1; SynB3; or any combination thereof,        or (iii) both (i) and (ii).        12. The method of item 10 or 11, wherein said peptide comprises:    -   (a) an ELD comprising the amino acid sequence of any one of SEQ        ID NOs: 1-15, 63, or 64, or a variant or fragment thereof having        endosomolytic activity;    -   (b) a CPD comprising the amino acid sequence of any one of SEQ        ID NOs: 16-27 or 65, or a variant or fragment thereof having        cell penetrating activity; or    -   (c) both (a) and (b).        13. The method of any one of items 10 to 12, wherein:    -   (i) said peptide comprises an ELD which is CM18, KALA, or        C(LLKK)₃C having the amino acid sequence of SEQ ID NO: 1, 14, or        63, or a variant thereof having at least 50%, 55%, 60%, 65%,        70%, 75%, 80%, 85%, 90%, or 95% identity to SEQ ID NO: 1, 14, or        63, and having endosomolytic activity;    -   (ii) wherein said peptide comprises a CPD which is TAT or PTD4        having the amino acid sequence of SEQ ID NO: 17 or 65, or a        variant thereof having at least 50%, 55%, 60%, 65%, 70%, 75%,        80%, 85%, 90%, or 95% identity to SEQ ID NO: 17 or 65 and having        cell penetrating activity; or    -   (iii) both (i) and (ii).        14. The method of any one of items 1 to 9, wherein said peptide        comprises the amino acid sequence of any one of SEQ ID NOs:        57-59, 66-72, or 82-102, or a functional variant thereof having        at least 85%, 90%, or 95% identity to any one of SEQ ID NOs:        57-59, 66-72, or 82-102.        15. The method of any one of items 1 to 14, wherein:    -   (i) said shuttle agent is completely metabolizable by the target        eukaryotic cell; and/or    -   (ii) contacting the target eukaryotic cell with the polypeptide        cargo in the presence of the shuttle agent at said concentration        results in an increase in the transduction efficiency of said        polypeptide cargo by at least 10-fold, 20-fold, 30-fold,        40-fold, 50-fold, or 100-fold, as compared to in the absence of        said shuttle agent.        16. The method of any one of items 1 to 15, which is an in vitro        method.        17. A synthetic peptide shuttle agent which is the peptide as        defined in any one of items 1 to 15.        18. The synthetic peptide of item 17, which is a peptide between        20 and 100 amino acids in length comprising the amino acid        sequence of any one of SEQ ID NOs: 104, 105, 107, 108, 110, 111,        112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124,        125, 126, 127, 128, 129, 130, 131, 133, 134, 135, 138, 140, 142,        145, 148, 151, 152, 169-242, and 243-10 242; or comprises the        amino acid sequence motifs of SEQ ID NOs: 158 and/or 159.        19. The synthetic peptide shuttle agent of item 17 or 18 for use        in delivering a polypeptide cargo from an extracellular space to        the cytosol and/or nucleus of a target eukaryotic cell in vitro,        wherein the synthetic peptide shuttle agent is used at a        concentration sufficient to increase the transduction efficiency        of said polypeptide cargo, as compared to in the absence of said        synthetic peptide shuttle agent.        20. The synthetic peptide shuttle agent of item 17 or 18 for use        in delivering a polypeptide cargo from an extracellular space to        the cytosol and/or nucleus of a target eukaryotic cell in vivo,        wherein the synthetic peptide shuttle agent is used at a        concentration sufficient to increase the transduction efficiency        of said polypeptide cargo, as compared to in the absence of said        synthetic peptide shuttle agent.        21. A composition comprising the shuttle agent as defined in any        one of items 1 to 15, or a cocktail of at least 2, at least 3,        at least 4, or at least 5 different types of the shuttle agents        as defined in any one of items 1 to 15, and a polypeptide cargo        to be delivered from an extracellular space to the cytosol        and/or nucleus of a target eukaryotic cell.        22. Use of the shuttle agent as defined in any one of items 1 to        15, or the synthetic peptide as defined in item 18, for        delivering a polypeptide cargo from an extracellular space to        the cytosol and/or nucleus of a target eukaryotic cell, wherein        the shuttle agent or synthetic peptide is used at a        concentration sufficient to increase the transduction efficiency        of said polypeptide cargo, as compared to in the absence of said        shuttle agent or synthetic peptide.        23. A kit for delivering a polypeptide cargo from an        extracellular space to the cytosol and/or nucleus of a target        eukaryotic cell, said kit comprising the shuttle agent as        defined in any one of items 1 to 15, or the synthetic peptide as        defined in item 18, and a suitable container.        24. The method of any one of items 1 to 16, the synthetic        peptide shuttle agent of any one of items 17 to 20, the        composition of item 21, the use of item 22, or the kit of item        23, wherein said polypeptide cargo lacks a cell penetrating        domain.        25. The method of any one of items 1 to 16, the synthetic        peptide shuttle agent of any one of items 17 to 20, the        composition of item 21, the use of item 22, or the kit of item        23, wherein said polypeptide cargo comprises a cell penetrating        domain.        26. The method of any one of items 1 to 16, 24 or 25, the        synthetic peptide shuttle agent of any one of items 17 to 20, 24        or 25, the composition of any one of items 21, 24 or 25, the use        of any one of items 22 to 25, wherein said polypeptide cargo        comprises a subcellular targeting domain.        27. The method, the synthetic peptide shuttle agent,        composition, use, or kit of item 26, wherein said subcellular        targeting domain is:    -   (a) a nuclear localization signal (NLS);    -   (b) a nucleolar signal sequence;    -   (c) a mitochondrial signal sequence; or    -   (d) a peroxisome signal sequence.        28. The method, the synthetic peptide shuttle agent,        composition, use, or kit of item 27, wherein:    -   (a) said NLS is from: E1a, T-Ag, c-myc, T-Ag, op-T-NLS, Vp3,        nucleoplasmin, histone 2B, Xenopus N1, PARP, PDX-1, QKI-5, HCDA,        H2B, v-Rel, Amida, RanBP3, Pho4p, LEF-1, TCF-1, BDV-P, TR2,        SOX9, or Max;    -   (b) said nucleolar signal sequence is from BIRC5 or RECQL4;    -   (c) said mitochondrial signal sequence is from Tim9 or Yeast        cytochrome c oxidase subunit IV; or    -   (d) said peroxisome signal sequence is from PTS1.        29. The method, the synthetic peptide shuttle agent,        composition, use, or kit of any one of items 24 to 29, wherein        said polypeptide cargo is complexed with a DNA and/or RNA        molecule.        30. The method, the synthetic peptide shuttle agent,        composition, use, or kit of any one of items 24 to 29, wherein        said polypeptide cargo is a transcription factor, a nuclease, a        cytokine, a hormone, a growth factor, an antibody, a peptide        cargo, an enzyme, an enzyme inhibitor, or any combination        thereof.        31. The method, the synthetic peptide shuttle agent,        composition, use, or kit of item 30, wherein:    -   (a) said transcription factor is: HOXB4, NUP98-HOXA9, Oct3/4,        Sox2, Sox9, Klf4, c-Myc, MyoD, Pdx1, Ngn3, MafA, Blimp-1, Eomes,        T-bet, FOXO3A, NF-YA, SALL4, ISL1, FoxA1, Nanog, Esrrb, Lin28,        HIF1-alpha, Hlf, Runx1t1, Pbx1, Lmo2, Zfp37, Prdm5, Bc-6, or any        combination thereof;    -   (b) said nuclease is a catalytically active or catalytically        dead: RNA-guided endonuclease, CRISPR endonuclease, type I        CRISPR endonuclease, type II CRISPR endonuclease, type III        CRISPR endonuclease, type IV CRISPR endonuclease, type V CRISPR        endonuclease, type VI CRISPR endonuclease, CRISPR associated        protein 9 (Cas9), Cpf1, CasY, CasX, zinc-finger nuclease (ZFNs),        Transcription activator-like effector nucleases (TALENs), homing        endonuclease, meganuclease, DNA-guided nuclease,        Natronobacterium gregoryi Argonaute (NgAgo), or any combination        thereof;    -   (c) said antibody recognizes an intracellular antigen; and/or    -   (d) said peptide cargo recognizes an intracellular molecule.        32. The method, the synthetic peptide shuttle agent,        composition, use, or kit of any one of items 24 to 31, for use        in cell therapy, genome editing, adoptive cell transfer, and/or        regenerative medicine.        33. The method, the synthetic peptide shuttle agent,        composition, use, or kit of any one of items 24 to 32, wherein        said target eukaryotic cell is an animal cell, a mammalian cell,        a human cell, a stem cell, a primary cell, an immune cell, a T        cell, an NK cell, or a dendritic cell.        34. A eukaryotic cell comprising the shuttle agent as defined in        any one of items 1 to 15, the synthetic peptide shuttle agent as        defined in item 18, or the composition as defined in item 21.        35. The eukaryotic cell of item 34, which is an mammalian cell,        a mammalian cell, a human cell, a stem cell, a primary cell, an        immune cell, a T cell, an NK cell, or a dendritic cell.        36. A method for delivering one or more CRISPR-associated        endonucleases alone or with one or more corresponding guide RNA        and/or linear DNA templates, to a target eukaryotic cell, said        method comprising contacting the target eukaryotic cell with the        endonuclease in the presence of a shuttle agent at a        concentration sufficient to increase the transduction efficiency        of said endonuclease, as compared to in the absence of said        shuttle agent, wherein said shuttle agent is as defined in any        one of items 1 to 15.        37. The method of item 36, which is an in vitro method, or an in        vivo method.        38. The method of item 36 or 37, wherein said one or more        endonuclease is: a type I CRISPR endonuclease, a type II CRISPR        endonuclease, a type III CRISPR endonuclease, a type IV CRISPR        endonuclease, a type V CRISPR endonuclease, a type VI CRISPR        endonuclease, or any combination thereof.        39. The method of item 36 or 37, wherein said one or more        endonuclease is CRISPR associated protein 9 (Cas9), Cpf1, CasX,        CasY, or any combination thereof; or a catalytically dead CRISPR        associated protein 9 (dCas9), dCpf1, dCasX, dCasY, or any        combination thereof.        40. The method of any one of items 36 to 39, wherein said target        eukaryotic cell is an animal cell, a mammalian cell, a human        cell, a stem cell, a primary cell, an immune cell, a T cell, an        NK cell, or a dendritic cell.        41. The method of item 58, wherein said one or more        corresponding guide RNA and/or linear DNA template targets one        or more genes to reduce the immunogenicity, improve        cytotoxicity, and/or otherwise improve the effectiveness of the        target eukaryotic cell for cell-based therapy, as compared to a        corresponding parent eukaryotic cell that has not been subjected        to said method.        42. The method of item 41, wherein said cell-based therapy is        cell-based cancer immunotherapy.        43. The method of any one of items 40 to 42, wherein said one or        more corresponding guide RNA and/or linear DNA template targets        the CBLB gene, c-CBL gene, GSK3 gene, ILT2 gene, CISH gene,        NKG2a gene, B2M gene, or any combination thereof.        44. A method for producing a synthetic peptide shuttle agent        that delivers a polypeptide cargo from an extracellular space to        the cytosol and/or nucleus of a target eukaryotic cell, said        method comprising synthesizing a peptide which is:    -   (1) a peptide at least 20 amino acids in length comprising    -   (2) an amphipathic alpha-helical motif having    -   (3) a positively-charged hydrophilic outer face, and a        hydrophobic outer face,        wherein at least five of the following parameters (4) to (15)        are respected:    -   (4) the hydrophobic outer face comprises a highly hydrophobic        core consisting of spatially adjacent L, I, F, V, W, and/or M        amino acids representing 12 to 50% of the amino acids of the        peptide, based on an open cylindrical representation of the        alpha-helix having 3.6 residues per turn;    -   (5) the peptide has a hydrophobic moment (μ) of 3.5 to 11;    -   (6) the peptide has a predicted net charge of at least +4 at        physiological pH;    -   (7) the peptide has an isoelectric point (pI) of 8 to 13;    -   (8) the peptide is composed of 35% to 65% of any combination of        the amino acids A, C, G, I, L, M, F, P, W, Y, and V;    -   (9) the peptide is composed of 0% to 30% of any combination of        the amino acids: N, Q, S, and T;    -   (10) the peptide is composed of 35% to 85% of any combination of        the amino acids A, L, K, or R;    -   (11) the peptide is composed of 15% to 45% of any combination of        the amino acids: A and L, provided there being at least 5% of L        in the peptide;    -   (12) the peptide is composed of 20% to 45% of any combination of        the amino acids K and R;    -   (13) the peptide is composed of 0% to 10% of any combination of        the amino acids: D and E;    -   (14) the difference between the percentage of A and L residues        in the peptide (% A+L), and the percentage of K and R residues        in the peptide (K+R), is less than or equal to 10%; and    -   (15) the peptide is composed of 10% to 45% of any combination of        the amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T,        and H.        45. The method of item 44, wherein the peptide is as defined in        any one of items 2 to 15.        46. A method for identifying a shuttle agent that delivers a        polypeptide cargo from an extracellular space to the cytosol        and/or nucleus of a target eukaryotic cell, said method        comprising:    -   (a) synthesizing a peptide which is the peptide as defined in        any one items 1 to 15 or 18;    -   (b) contacting the target eukaryotic cell with the polypeptide        cargo in the presence of said peptide;    -   (c) measuring the transduction efficiency of the polypeptide        cargo in the target eukaryotic cell; and    -   (d) identifying the peptide as being a shuttle agent that        transduces the polypeptide cargo, when an increase in the        transduction efficiency of said polypeptide cargo in the target        eukaryotic cell is observed.        47. The method of item 46, wherein said polypeptide cargo is as        defined in any one of items 24 to 31.        48. A genome editing system comprising:    -   (a) the shuttle agent as defined in any one items 1 to 15 or 18;    -   (b) one or more CRISPR-associated endonucleases; and    -   (c) one or more guide RNAs.        49. The genome editing system of item 48, further comprising a        linear DNA template for controlling the genome editing.        50. The genome editing system of item 48 or 49, wherein said one        or more CRISPR-associated endonucleases is: a type I CRISPR        endonuclease, a type II CRISPR endonuclease, a type III CRISPR        endonuclease, a type IV CRISPR endonuclease, a type V CRISPR        endonuclease, a type VI CRISPR endonuclease, CRISPR associated        protein 9 (Cas9), Cpf1, CasX, CasY, or any combination thereof.        51. A method for enriching eukaryotic cells transduced with a        polypeptide cargo of interest, said method comprising:    -   (a) co-transducing a target eukaryotic cell population with a        polypeptide cargo of interest and a marker protein; and    -   (b) isolating or concentrating eukaryotic cells transduced with        the marker protein, thereby enriching eukaryotic cells        transduced with the polypeptide cargo of interest.        52. The method of item 51, wherein:    -   (i) the marker protein is not covalently bound to the        polypeptide cargo of interest, the marker protein is covalently        bound to the polypeptide cargo of interest, the marker protein        is non-covalently bound to the polypeptide cargo of interest, or        the marker protein is covalently bound to the polypeptide cargo        of interest via a cleavable linker; and/or    -   (ii) the marker protein comprises a detectable label, or the        marker protein is a fluorescent protein, a fluorescently-labeled        protein, a bioluminescent protein, an isotopically-labelled        protein, or a magnetically-labeled protein.        53. The method of item 51 or 52, wherein the intracellular        concentration of the transduced marker protein is positively        correlated with the intracellular concentration of the        transduced polypeptide cargo of interest.        54. The method of any one of items 51 to 53, wherein the        eukaryotic cells transduced with the marker protein are isolated        or concentrated using flow cytometry, fluorescence-activated        cell sorting (FACS), or magnetic-activated cell sorting (MACS).        55. The method of any one of items 51 to 54, wherein the        eukaryotic cells transduced with the marker protein are isolated        or sorted from cells lacking the marker protein, thereby        producing a marker protein-positive cell population and/or a        marker protein-negative cell population.        56. The method of item 55, further comprising repeating        steps (a) and (b), one or more times, on the marker        protein-negative cell population, on the marker protein-positive        cell population, or on both the marker protein-negative and the        marker protein-positive cell populations.        57. The method of any one of items 51 to 56, wherein the        eukaryotic cells transduced with the marker protein are isolated        or sorted based on their intracellular concentration of the        marker protein.        58. The method of any one of items 51 to 57, wherein the marker        protein is a protein that stimulates cell proliferation, a        protein that stimulates cell differentiation, a protein that        promotes cell survival, an anti-apoptotic protein, or a protein        having another biological activity.        59. The method of any one of items 51 to 58, wherein the        polypeptide cargo of interest and the marker protein are        co-transduced by contacting the target eukaryotic cell with the        polypeptide cargo and the marker protein in the presence of a        peptide transduction agent, wherein the peptide transduction        agent is present at a concentration sufficient to increase the        transduction efficiency of the polypeptide cargo and the marker        protein, as compared to in the absence of said peptide        transduction agent.        60. The method of item 59, wherein:    -   (a) the peptide transduction agent is an endosomolytic peptide;    -   (b) the peptide transduction agent is or comprises the synthetic        peptide shuttle agent as defined in item 17 or 18;    -   (c) the target eukaryotic cells comprise animal cells, mammalian        cells, human cells, stem cells, primary cells, immune cells, T        cells, NK cells, or dendritic cells;    -   (d) the polypeptide cargo of interest is: (i) the polypeptide        cargo as defined in any one of items 24 to 31; and/or (ii) one        or more CRISPR-associated endonucleases alone or with one or        more corresponding guide RNA and/or linear DNA templates as        defined in any one of items 38 to 43; or    -   (e) any combination of (a) to (d).

Other objects, advantages and features of the present description willbecome more apparent upon reading of the following non-restrictivedescription of specific embodiments thereof, given by way of exampleonly with reference to the accompanying drawings.

EXAMPLES Example 1 Materials and Methods 1.1 Materials

All chemicals were purchased from Sigma-Aldrich (St. Louis, Mo., USA orOakville, ON, Canada) or equivalent grade from BioShop Canada Inc.(Mississauga, ON, Canada) or VWR (Ville Mont-Royal, QC, Canada), unlessotherwise noted.

1.2 Reagents

TABLE 1.1 Reagents Material Company City, Province-State, Country RPMI1640 media Sigma-Aldrich Oakville, ON, Canada DMEM Sigma-AldrichOakville, ON, Canada Alpha MEM Stem Cell Technology Oakville, ON, CanadaFetal bovine serum (FBS) NorthBio Toronto, ON, Canada Horse serumInvitrogen Burlington, ON, Canada L-glutamine-Penicillin-StreptomycinSigma-Aldrich Oakville, ON, Canada Trypsin-EDTA solution Sigma-AldrichOakville, ON, Canada Inositol Sigma-Aldrich Oakville, ON, Canada Folicacid Sigma-Aldrich Oakville, ON, Canada pEGFP-C1 CLONTECH LaboratoriesPalo Alto, CA, USA FITC-Antibody α-tubulin Abcam ab64503 Cambridge, MA,USA ITS Invitrogen/41400-045 Burlington, ON, Canada FGF 2 FeldanBio/1D-07-017 Quebec, QC, Canada Dexamethasone Sigma-Aldrich/D8893Oakville, ON, Canada Bovine serum albumin (BSA) Sigma-Aldrich/A-1933Oakville, ON, Canada MB1 media GE Healthcare HyClone Logan, Utah, USACalcein Sigma-Aldrich/C0875 Oakville, ON, Canada HisTrap ™ FF column GEHealthcare Baie d'Urfe, QC, Canada Q Sepharose ™ GE Healthcare Baied'Urfe, QC, Canada SP Sepharose ™ GE Healthcare Baie d'Urfe, QC, CanadaAmicon Ultra centrifugal filters EMD Millipore Etobicoke, ON CanadaLabel IT ® Cy ®5 kit Mirus Bio LLC Madison, WI, USA Calf serum NorthBioToronto, ON, Canada beta-mercaptoethanol Sigma-Aldrich or Gibco-Oakville, ON, Canada ThermoFisher IL-2 Feldan Bio/rhIL-2 ResearchQuebec, QC, Canada Resazurin sodium salt Sigma-Aldrich/R7017-1GOakville, ON, Canada Anti-HOXB4 monoclonal antibody Novus Bio#NBP2-37257 Oakville, ON, Canada Alexa ™-594 Anti-Mouse Abcam #150116Toronto, ON, Canada Fluoroshield ™ with DAPI Sigma #F6057 Oakville, ON,Canada GFP Monoclonal antibody Feldan Bio #A017 Quebec, QC, CanadaPhusion ™ High-Fidelity DNA polymerase (NEB #M0530S) Whitby, ON, CanadaEdit-R ™ Synthetic crRNA Positive (Dharmacon #U-007000-05) Ottawa, ON,Canada Controls T7 Endonuclease I (NEB, Cat #M0302S) Whitby, ON, CanadaFastFect ™ transfection reagent (Feldan Bio #9K-010-0001) Quebec, QC,Canada Goat Anti-Mouse IgG H&L (Alexa Fluor ® Abcam ab150113 Toronto,ON, Canada 488) Goat Anti-Rabbit IgG H&L Abcam ab150080 Toronto, ON,Canada (Alexa Fluor ® 594) Opti-MEM ™ Sigma-Aldrich Oakville, ON, CanadaAnti-NUP98 Abcam #ab50610 Toronto, ON, Canada PARP (Cleaved) [214/215]Human ELISA ThermoFisher #KHO0741 Burlington, ON, Canada Kit PurifiedRabbit Anti-Active Caspase-3 BD Biosciences #559565 Mississauga, ON,Canada Active Caspace-3 antibody Cedarlane #AF835 Burlington, ON, CanadaTNF-alpha Antibody BioVision Inc #3054-100 Milpitas, CA, USA APC MouseAnti-Human HLA-ABC BD Biosciences #555555 Mississauga, ON, CanadaAnti-CD3 Biolegend#cat: 300438 San Diego, CA, USA Anti-CD28 Thermofisher#cat: 16-0289-85 Burlington, ON, Canada

1.3 Cell Lines

HeLa, HEK293A, HEK293T, THP-1, CHO, NIH3T3, CA46, Balb3T3, HT2, KMS-12,DOHH2, REC-1, HCC-78, NCI-H196 and HT2 cells were obtained from AmericanType Culture Collection (Manassas, Va., USA) and cultured following themanufacturer's instructions. Myoblasts are primary human cells kindlyprovided by Professor J. P. Tremblay (Université Laval, Quebec, Canada).

TABLE 1.2 Cell lines and culture conditions Culture Cell linesDescription ATCC/others media Serum Additives HeLa Human cervical ATCC ™CCL-2 DMEM 10% FBS L-glutamine 2 mM (adherent carcinoma cells Penicillin100 units cells) Streptomycin 100 μg/mL HEK 293A Human embryonic ATCC ™CRL-1573 DMEM 10% FBS L-glutamine 2 mM (adherent Epithelial kidneyPenicillin 100 units cells) cells Streptomycin 100 μg/mL HEK 293T Humanembryonic ATCC ™ CRL-3216 DMEM 10% FBS L-glutamine 2 mM (adherentEpithelial kidney Penicillin 100 units cells) cells Streptomycin 100μg/mL THP-1 Acute monocytic ATCC ™ TIB202 RPMI 1640 10% FBSβ-mercaptoethanol 0.05 mM (suspension leukemia L-glutamine 2 mM cells)Penicillin 100 units Streptomycin 100 μg/mL Myoblasts Human (13 months)Kindly provided by MB1 15% FBS ITS 1x, FGF 2 10 ng/mL, (primarymyoblasts Professor J. P. Dexamethasone 0.39 μg/mL, adherent TremblayBSA 0.5 mg/mL, cells) MB1 85% CHO Chinese hamster ATCC ™ CCL-61 DMEM 10%FBS L-glutamine 2 mM (adherent ovary cells Penicillin 100 units cells)Streptomycin 100 μg/mL NIH3T3 Fibroblasts ATCC ™ CRL-1658 DMEM 10% CalfL-glutamine 2 mM (adherent serum Penicillin 100 units cells)Streptomycin 100 μg/mL HT2 T lymphocytes ATCC ™ CRL-1841 RPMI 1640 10%FBS 200 IU/mL IL-2 (suspension β-mercaptoethanol 0.05 mM cells)L-glutamine 2 mM Penicillin 100 units Streptomycin 100 μg/mL CA46 Homosapiens ATCC ™ CRL-1648 RPMI 1640 20% FBS L-glutamine 2 mM (suspensionBurkitt's lymphoma Penicillin 100 units cells) Streptomycin 100 μg/mLBalb3T3 Fibroblasts ATCC ™ CCL-163 DMEM 10% Calf L-glutamine 2 mM(adherent serum Penicillin 100 units cells) Streptomycin 100 μg/mLJurkat Human T cells ATCC ™ TIB-152 RPMI 1640 10% FBS L-glutamine 2 mM(suspension Penicillin 100 units cells) Streptomycin 100 μg/mL DOHH2Human B cell Gift from RPMI 1640 10% FBS L-glutamine 2 mM (suspensionlymphoma Horizon Inc. Penicillin 100 units cells) Streptomycin 100 μg/mLKMS-12 Myeloma bone Gift from Advanced 10% FBS L-glutamine 2 mM(suspension marrow Horizon Inc. RPMI 1640 Penicillin 100 units cells)Streptomycin 100 μg/mL REC-1 Human lymph node Gift from RPMI 1640 10%FBS L-glutamine 2 mM (suspension mantel cell Horizon Inc. Penicillin 100units cells) Streptomycin 100 μg/mL HCC-78 Human Gift from RPMI 1640 20%FBS L-glutamine 2 mM (adherent adenocarcinoma Horizon Inc. Penicillin100 units cells) lung cell Streptomycin 100 μg/mL NCI-H196 Human smallcell Gift from RPMI 1640 10% FBS L-glutamine 2 mM (adherent lung cancerHorizon Inc. Penicillin 100 units cells) Streptomycin 100 μg/mL NK Humannormal All cells ™ RPMI 1640 10% FBS 200 IU/mL IL-2 (suspensionPeripheral Blood #PB012-PF L-glutamine 2 mM cells) CD56+ lymphocytePenicillin 100 units Streptomycin 100 μg/mL NK-92 Human normal Gift fromCETC Alpha MEM 12.5% FBS L-glutamine 200 nM (suspension Peripheral Blood12.5% Horse IL-2 25000 U/mL cells) CD56+ lymphocyte serum Inositol 1MFolic acid 100 nM B-ME 55 mM T cells Human normal Healthy blood RPMI 10%FBS L-glutamine 200 nM (suspension Peripheral Blood donor advanced IL-225000 U/mL cells) CD3+ lymphocyte Penicillin 100 units Streptomycin 100μg/mL FBS: Fetal bovine serum

1.4 Protein Purification

Fusion proteins were expressed in bacteria (E. coli BL21DE3) understandard conditions using an isopropyl β-D-1-thiogalactopyranoside(IPTG) inducible vector containing a T5 promoter. Culture mediacontained 24 g yeast extract, 12 g tryptone, 4 mL glycerol, 2.3 gKH₂PO₄, and 12.5 g K₂HPO₄ per liter. Bacterial broth was incubated at37° C. under agitation with appropriate antibiotic (e.g., ampicillin).Expression was induced at optical density (600 nm) between 0.5 and 0.6with a final concentration of 1 mM IPTG for 3 hours at 30° C. Bacteriawere recuperated following centrifugation at 5000 RPM and bacterialpellets were stored at −20° C.

Bacterial pellets were resuspended in Tris buffer (Tris 25 mM pH 7.5,NaCl 100 mM, imidazole 5 mM) with phenylmethylsulfonyl fluoride (PMSF) 1mM, and lysed by passing 3 times through the homogenizer Panda 2K™ at1000 bar. The solution was centrifuged at 15000 RPM, 4° C. for 30minutes. Supernatants were collected and filtered with a 0.22 μMfiltration device.

Solubilized proteins were loaded, using a FPLC (AKTA Explorer 100R), onHisTrap™ FF column previously equilibrated with 5 column volumes (CV) ofTris buffer. The column was washed with 30 column volumes (CV) of Trisbuffer supplemented with 0.1% Triton™ X-114 followed with 30 CV of Trisbuffer with imidazole 40 mM. Proteins were eluted with 5 CV of Trisbuffer with 350 mM Imidazole and collected. Collected fractionscorresponding to specific proteins were determined by standarddenaturing SDS-PAGE.

Purified proteins were diluted in Tris 20 mM at the desired pH accordingto the protein's pI and loaded on an appropriate ion exchange column (QSepharose™ or SP Sepharose™) previously equilibrated with 5 CV of Tris20 mM, NaCl 30 mM. The column was washed with 10 CV of Tris 20 mM, NaCl30 mM and proteins were eluted with a NaCl gradient until 1 M on 15 CV.Collected fractions corresponding to specific proteins were determinedby standard denaturing SDS-PAGE. Purified proteins were then washed andconcentrated in PBS 1× on Amicon Ultra™ centrifugal filters 10,000 MWCO.Protein concentration was evaluated using a standard Bradford assay.

1.5 Synthetic Peptides and Shuttle Agents

All peptides used in this study were purchased from GLBiochem (Shanghai,China) and their purities were confirmed by high-performance liquidchromatography analysis and mass spectroscopy. In some cases, peptideswere synthesized to contain a C-terminal cysteine residue to allow thepreparation of peptide dimers. These dimeric peptides were directlysynthesized with a disulfide bridge between the C-terminal cysteines oftwo monomers. The amino acid sequences and characteristics of each ofthe synthetic peptides and shuttle agents tested in the present examplesare summarized in Table 1.3, Table B1, and Table C1.

TABLE 1.3 Synthetic peptides and shuttle agentsAmino acid (a.a.) sequence Hydro- [SEQ ID NO; not including pathi-Peptide or C-terminal Cys, unless MW city Domain(s) Shuttle agentindicated with an *] a.a. (kDa) pl Charge index ELD CM18KWKLFKKIGAVLKVLTTG [1] 18 2.03 10.60  5+/0−  0.350 C(LLKK)₃CCLLKKLLKKLLKKC [63] 14 1.69 10.05  6+/0−  0.314 LAH4KKALLALALHHLAHLALHLALALKKA [6] 26 2.78 10.48  4+/0−  0.923 KALAWEAKLAKALAKALAKHLAKALAKALKACEA 30 3.13 9.9  7+/2−  0.283 [14] CPDTAT-cys YGRKKRRQRRRC [17] 12 1.66 12.01  8+/0− −3.125 Penetratin-cysRQIKIWFQNRRMKWKKC [18] 17 2.35 11.75  7+/0− −1.482 PTD4 YARAAARQARA [65]11 1.2 11.72  3+/0− −0.682 His- His-PTD4 HHHHHHYARAAARQARA [81] 17 2.0311.71  3+/0− −1.57  PTD4 CPD-ELD TAT-CM18 YGRKKRRQRRRCKWKLFKKIGAVLKVLTTG30 3.68 12.02 13+/0− −1.041 [66] TAT-KALA YGRKKRRQRRRCWEAKLAKALAKALAKHL42 4.67 11.46 15+/2− −0.768 AKALAKALKACEA [67] PTD4-KALAYARAAARQARAWEAKLAKALAKALAKHLAK 41 4.32 10.46 10+/2−  0.024ALAKALKACEA [82] 9Arg-KALA RRRRRRRRRWEAKLAKALAKALAKHLAKAL 39 4.54 12.1116+/2− −0.821 AKALKACEA [83] Pep1-KALA KETWWETWWTEWSQPKKKRKVWEAKLAK 515.62 10.01 13+/5− −0.673 ALAKALAKHLAKALAKALKACEA [84] Xentry-KALALCLRPVGWEAKLAKALAKALAKHLAKALAK 37 3.87  9.93  8+/2−  0.441 ALKACEA[85]SynB3-KALA RRLSYSRRRFWEAKLAKALAKALAKHLAKA 40 4.51 11.12 12+/2− −0.258LAKALKACEA[86] ELD-CPD CM18-TAT-Cys KWKLFKKIGAVLKVLTTGYGRKKRRQRRRC 303.67 12.02 13+/0− −1.04  [57] CM18-Penetratin-KWKLEKKIGAVLKVLTTGRQIKIWFQNRRMK 35 4.36 11.36 12+/0− −0.54  CysWKKC [58] dCM18-TAT-Cys KWKLFKKIGAVLKVLTTGYGRKKRRQRRRC 60 7.34 12.1626+/0− −1.04  (CM18-TAT-cys [57] dimer) KWKLFKKIGAVLKVLTTGYGRKKRRQRRRC[57] dCM18-Penetratin- KWKLFKKIGAVLKVLTTGRQIKIWFQNRRMK 70 8.72 12.0524+/0− −0.54  Cys WKKC [58] (CM18-Penetratin-KWKLFKKIGAVLKVLTTGRQIKIWFQNRRMK Cys dimer) WKKC [58] VSVG-PTD4KFTIVFPHNQKGNWKNVPSNYHYCPYARA 36 4.2 10.3   6+/0− −0.89  AARQARA [87]EB1-PTD4 LIRLWSHLIHIWFQNRRLKWKKKYARAAAR 34 4.29 12.31 10+/0− −0.647QARA [88] JST-PTD4 GLFEALLELLESLWELLLEAYARAAARQARA 31 3.49  4.65  5+/3− 0.435 [89] CM18-PTD4 KWKLFKKIGAVLKVLTTGYARAAARQARA 29 3.217 11.76 8+/0− −0.041 [90] 6Cys-CM18-PTD4 CCCCCCKWKLFKKIGAVLKVLTTGYARAAA 353.835 9.7  8+/0−  0.394 RQARA [91] CM18-L1-PTD4KWKLFKKIGAVLKVLTTGGGSYARAAARQA 32 3.42 11.76  8+/0− −0.087 RA [92]CM18-L2-PTD4 KWKLFKKIGAVLKVLTTGGGSGGGSYARAA 36 3.68 11.76  8+/0− −0.133ARQARA [93] CM18-L3-PTD4 KWKLFKKIGAVLKVLTTGGGSGGGSGGGS 41 3.99 11.76 8+/0− −0.176 GYARAAARQARA [94] His-ELD- Met-His-CM18-TAT-MHHHHHHKWKLFKKIGAVLKVLTTGYGRKK 37 4.63 12.02 13+/0− −1.311 CPD CysRRQRRRC [59*] His-CM18-TAT HHHHHHKWKLFKKIGAVLKVLTTGYGRKKR 35 4.4 12.3113+/0− −1.208 RQRRR [95] His-CM18-PTD4 HHHHHHKWKLFKKIGAVLKVLTTCYARAAA 354.039 11.76  8+/0− −0.583 RQARA [68] His-CM18-PTD4-HHHHHHKWKLFKKIGAVLKVLTTGYARAAA 41 4.659 9.7  8+/0− −0.132 6CysRQARACCCCCC [96*] His-CM18-9Arg HHHHHHKWKLFKKIGAVLKVLTTGRRRRRR 33 4.2612.91 14+/0− −1.618 RRR [69] His-CM18- HHHHHHKWKLFKKIGAVLKVLTTGGWTLNS 505.62 10.6   9+/0−  0.092 Transportan AGYLLKINLKALAALAKKIL [70] His--PTD4HHHHHHKKALLALALHHLAHLALHLALALKK 43 4.78 11.75  7+/0− −0.63 AYARAAARQARA [71] His-C(LLKK)₃C- HHHHHHCLLKKLLKKLLKKCYARAAARQAR 31 3.5611.21  9+/0− −0.827 PTD4 A [72] 3His-CM18-PTD4HHHKWKLFKKIGAVLKVLTTGYARAAARQA 32 3.63 11.76  8+/0− −0.338 RA [97]12His-CM18-PTD4 HHHHHHHHHHHHKWKLFKKIGAVLKVLTTG 41 4.86 11.78  8+/0−−0.968 YARAAARQARA [98] HA-CM18-PTD4 HHHAHHHKWKLFKKIGAVLKVLTTGYARAA 364.11 11.76  8+/0− −0.517 ARQARA [99] 3HA-CM18-PTD4HAHHAHHAHKWKLFKKIGAVLKVLTTGYAR 38 4.25 11.78  8+/0− −0.395AAARQARA [100] ELD-His- CM18-His-PTD4 KWKLFKKIGAVLKVLTTGHHHHHHYARAAA 354.04 11.76  8+/0− −0.583 CPD RQARA [101] His-ELD- His-CM18-PTD4-HHHHHHKWKLFKKIGAVLKVLTTGYARAAA 41 4.86 11.76  8+/0− −0.966 CPD-His HisRQARAHHHHHH [102] Results computed using the ProtParam ™ online toolavailable from ExPASy ™ Bioinformatics Resource Portal(http://web.expasy.org/protparam/) MW: Molecular weight pl: Isoelectricpoint Charge: Total number of positively (+) and negatively (−) chargedresidues

Example 2 Peptide Shuttle Agents Facilitate Escape ofEndosomally-Trapped Calcein 2.1 Endosome Escape Assays

Microscopy-based and flow cytometry-based fluorescence assays weredeveloped to study endosome leakage and to determine whether theaddition of the shuttle agents facilitates endosome leakage of thepolypeptide cargo. These methods are described in Example 2 ofPCT/CA2016/050403.

2.1.1 Endosomal Leakage Visualization by Microscopy

Calcein is a membrane-impermeable fluorescent molecule that is readilyinternalized by cells when administered to the extracellular medium. Itsfluorescence is pH-dependent and calcein self-quenches at higherconcentrations. Once internalized, calcein becomes sequestered at highconcentrations in cell endosomes and can be visualized by fluorescencemicroscopy as a punctate pattern. Following endosomal leakage, calceinis released to the cell cytoplasm and this release can be visualized byfluorescence microscopy as a diffuse pattern.

One day before the calcein assay was performed, cells in exponentialgrowth phase were harvested and plated in a 24-well plate (80,000 cellsper well). The cells were allowed to attach by incubating overnight inappropriate growth media, as described in Example 1. The next day, themedia was removed and replaced with 300 μL of fresh media without FBScontaining 62.5 μg/mL (100 μM) of calcein, except for HEK293A (250μg/mL, 400 μM). At the same time, the shuttle agent(s) to be tested wasadded at a predetermined concentration. The plate was incubated at 37°C. for 30 minutes. The cells were washed with 1×PBS (37° C.) and freshmedia containing FBS was added. The plate was incubated at 37° C. for2.5 hours. The cells were washed three times and were visualized byphase contrast and fluorescence microscopy (IX81™, Olympus).

A typical result is shown in FIG. 1A, in which untreated HEK293A cellsloaded with calcein (“100 μM calcein”) show a low intensity, punctatefluorescent pattern when visualized by fluorescence microscopy (upperleft panel). In contrast, HeLa cells treated with a shuttle agent thatfacilitates endosomal escape of calcein (“100 μM calcein+CM18-TAT 5 μM”)show a higher intensity, more diffuse fluorescence pattern in a greaterproportion of cells (upper right panel).

2.1.2 Endosomal Leakage Quantification by Flow Cytometry

In addition to microscopy, flow cytometry allows a more quantitativeanalysis of the endosomal leakage as the fluorescence intensity signalincreases once the calcein is released in the cytoplasm. Calceinfluorescence is optimal at physiological pH (e.g., in the cytosol), ascompared to the acidic environment of the endosome.

One day before the calcein assay was performed, cells in exponentialgrowth phase were harvested and plated in a 96-well plate (20,000 cellsper well). The cells were allowed to attach by incubating overnight inappropriate growth media, as described in Example 1. The next day, themedia in wells was removed and replaced with 50 μL of fresh mediawithout serum containing 62.5 μg/mL (100 μM) of calcein, except forHEK293A (250 μg/mL, 400 μM). At the same time, the shuttle agent(s) tobe tested was added at a predetermined concentration. The plate wasincubated at 37° C. for 30 minutes. The cells were washed with 1×PBS(37° C.) and fresh media containing 5-10% serum was added. The plate wasincubated at 37° C. for 2.5 hours. The cells were washed with 1×PBS anddetached using trypsinization. Trypsinization was stopped by addition ofappropriate growth media, and calcein fluorescence was quantified usingflow cytometry (Accuri C6, Becton, Dickinson and Company (BD)).

Untreated calcein-loaded cells were used as a control to distinguishcells having a baseline of fluorescence due to endosomally-trappedcalcein from cells having increased fluorescence due to release ofcalcein from endosomes. Fluorescence signal means (“mean counts”) wereanalyzed for endosomal escape quantification. In some cases, the “MeanFactor” was calculated, which corresponds to the fold-increase of themean counts relative to control (untreated calcein-loaded cells). Also,the events scanned by flow cytometry corresponding to cells (size andgranularity) were analyzed. The cellular mortality was monitored withthe percentage of cells in the total events scanned. When it becamelower than the control, it was considered that the number of cellulardebris was increasing due to toxicity and the assay was discarded.

A typical result is shown in FIG. 1B, in which an increase influorescence intensity (right-shift) is observed for calcein-loaded HeLacells treated with a shuttle agent that facilitates endosomal escape(“Calcein 100 μM+CM18-TAT 5 μM”, right panel), as compared to untreatedcalcein-loaded HeLa cells (“Calcein 100 μM”, left panel). The increasein calcein fluorescence is caused by the increase in pH associated withthe release of calcein from the endosome (acidic) to the cytoplasm(physiological).

2.2 Results from Endosome Escape Assays

2.2.1 HeLa Cells

HeLa cells were cultured and tested in the endosomal escape assays asdescribed in Example 2.1. The results of flow cytometry analyses aresummarized below. In each case, the flow cytometry results were alsoconfirmed by fluorescence microscopy (data not shown).

TABLE 2.1 CM18-Penetratin-Cys v. Controls in Hela cells Concen- trationMean Counts Mean Domains Peptide Cells (μm) (±St Dev.: n = 3) Factor —No peptide HeLa 0 55 359 ± 6844 1.0 ELD CM18 HeLa 5 46 564 ± 9618 0.8CPD TAT-Cys HeLa 5 74 961 ± 9337 1.3 Penetratin- HeLa 5 59 551 ± 71191.1 Cys ELD + CPD CM18 + HeLa 5 + 5 64 333 ± 6198 1.2 TAT-Cys CM18 +HeLa 5 + 5 40 976 ± 8167 0.7 Penetratin- Cys ELD − CPD CM18 − HeLa 5 262 066 ± 28 146 4.7 Penetratin- Cys

TABLE 2.2 CM18-TAT-Cys v. Control in HeLa cells Concentration Meancounts Mean Domains Peptide Cells (μM) (n = 3) Stand. dev. Factor — Nopeptide HeLa 0  53 369   4192 1.0 ELD-CPD CM18-TAT-Cys HeLa 5 306 572 46564 5.7

The results in Tables 2.1 and 2.2 show that treating calcein-loaded HeLacells with the shuttle agents CM18-Penetratin-Cys and CM18-TAT-Cys(having the domain structure ELD-CPD) results in increased mean cellularcalcein fluorescence intensity, as compared to untreated control cellsor cells treated with single-domain peptides used alone (CM18, TAT-Cys,Penetratin-Cys) or together (CM18+TAT-Cys, CM18+Penetratin-Cys). Theseresults suggest that CM18-Penetratin-Cys and CM18-TAT-Cys facilitateescape of endosomally-trapped calcein, but that single domain peptides(used alone or together) do not.

TABLE 2.3 Dose response of CM18-TAT-Cys in HeLa cells, data from FIG. 2Concentration Mean counts Domains Peptide Cells (μM) (n = 3) Stand. dev.Mean Factor — No peptide HeLa 0  63 872 11 587 1.0 (“calcein 100 μM”)ELD-CPD CM18-TAT-Cys HeLa 1  86 919 39 165 1.4 CM18-TAT-Cys HeLa 2 137887 13 119 2.2 CM18-TAT-Cys HeLa 3 174 327 11 519 2.7 CMI8-TAT-Cys HeLa4 290 548 16 593 4.5 CMI8-TAT-Cys HeLa 5 383 685   5578 6.0

TABLE 2.4 Dose response of CM18-TAT-Cys in HeLa cells Concentration Meancounts Domains Peptide Cells (μM) (n = 3) Stand. dev. Mean Factor — Nopeptide HeLa 0  81 013 14 213 1.0 ELD-CPD CM18-TAT-Cys HeLa 3 170 652 63848 2.1 CM18-TAT-Cys HeLa 4 251 799 33 880 3.1 CM18-TAT-Cys HeLa 5 335324 10 651 4.1

TABLE 2.5 Dose response of CM18-TAT-Cys and CM18-Penetratin-Cys in HeLacells, data from FIG. 3 Concentration Mean counts Domains Peptide Cells(μM) (n = 3) Stand. dev. Mean Factor — No peptide HeLa 0  62 503 23 7521.0 ELD-CPD CM18-TAT-Cys HeLa 5 187 180   8593 3.0 CM18-TAT-Cys HeLa 8321 873 36 512 5.1 CM18-Penetratin-Cys HeLa 5 134 506   2992 2.2CM18-Penetratin-Cys HeLa 8 174 233 56 922 2.8

The results in Tables 2.3 (FIG. 2), 2.4, and 2.5 (FIG. 3) suggest thatCM18-TAT-Cys and CM18-Penetratin-Cys facilitate escape ofendosomally-trapped calcein in HeLa cells in a dose-dependent manner. Insome cases, concentrations of CM18-TAT-Cys or CM18-Penetratin-Cys above10 μM were associated with an increase in cell toxicity in HeLa cells.

TABLE 2.6 Dimers v. monomers of CM18-TAT-Cys and CM18-Penetratin-Cys inHeLa cells Concentration Mean counts Domains Peptide Cells (μM) (n = 4)Stand. dev. Mean Factor — No peptide HeLa 0  60 239 9860 1.0 ELD-CPDCM18-TAT-Cys HeLa 4 128 461 25 742   2.1 CM18-Penetratin-Cys HeLa 4 116873 3543 1.9 ELD-CPD dCM18-TAT-Cys HeLa 2  79 380 4297 1.3 dimerdCM18-Penetratin-Cys HeLa 2 128 363 8754 2.1

TABLE 2.7 Monomers v. dimers of CM18-TAT-Cys and CM18-Penetratin-Cys inHeLa cells Con- Mean Mean centraton counts Stand. Fac- Domains PeptideCells (μM) (n = 3) dev. tor — No peptide HeLa 0  55 834 1336 1.0 ELD-CPDCM18-TAT- HeLa 4 159 042 16 867 2.8 Cys ELD-CPD dCM18-TAT- HeLa 2 174274  9 553 3.1 dimer Cys

The results in Table 2.6 and 2.7 suggest that shuttle peptide dimers(which are molecules comprising more than one ELD and CPD) are able tofacilitate calcein endosomal escape levels that are comparable to thecorresponding monomers.

2.2.3 HEK293A Cells

To examine the effects of the shuttle agents on a different cell line,HEK293A cells were cultured and tested in the endosomal escape assays asdescribed in Example 2.1. The results of flow cytometry analyses aresummarized below in Table 2.8 and in FIG. 1B.

TABLE 2.8 CM18-TAT-Cys in HEK293A cells Con- cen- Mean tration countsStand. Mean Domains Peptide Cells (μM) (n = 2) dev. Factor — No peptideHEK293A 0 165 819 7693 1.0 ELD- CM18-TAT- HEK293A 0.5 196 182 17 224 1.2CPD Cys CM18-TAT- HEK293A 5 629 783 1424 3.8 Cys

The results in Table 2.8 and in FIG. 1B show that treatingcalcein-loaded HEK293A cells with the shuttle agent CM18-TAT-Cys resultsin increased mean cellular calcein fluorescence intensity, as comparedto untreated control cells.

2.2.2 Myoblasts

To examine the effects of the shuttle agents on primary cells, primarymyoblast cells were cultured and tested in the endosomal escape assaysas described in Example 2.1. The results of flow cytometry analyses aresummarized below in Tables 2.9 and 2.10, and in FIG. 4. In each case,the flow cytometry results were also confirmed by fluorescencemicroscopy.

TABLE 2.9 Dose response of CM18-TAT-Cys in primary myoblasts, data fromFIG. 4 Peptide Mean Conc. counts Stand. Mean Domains Peptide Cells (μM)(n = 3) dev. Factor — No peptide; Myoblasts 0 863 61 n/a no calcein(“Cells”) — No peptide Myoblasts 0 38 111 13 715 1.0 (“Calcein 100 μM”)ELD- CM18-TAT- Myoblasts 5 79 826 12 050 2.1 CPD Cys CM18-TAT- Myoblasts8 91 421 10 846 2.4 Cys

TABLE 2.10 Dose response of CM18-TAT-Cys in primary myoblasts PeptideMean Conc. counts Stand. Mean Domains Peptide Cells (μM) (n = 3) dev.Factor — No peptide Myoblasts 0 31 071 21 075 1.0 ELD- CM18-TAT-Myoblasts 5 91 618 10 535 2.9 CPD Cys CM18-TAT- Myoblasts 7.5 95 289 11266 3.1 Cys

The results in Table 2.9 (shown graphically in FIG. 4) and Table 2.10suggest that CM18-TAT-Cys facilitates escape of endosomally-trappedcalcein in a dose-dependent manner in primary myoblasts. Concentrationsof CM18-TAT-Cys above 10 μM were associated with an increase in celltoxicity in myoblast cells, as for HeLa cells.

TABLE 2.11 Monomers v. dimers CM18-TAT-Cys and CM18-Penetratin-Cys inprimary myoblasts Con- cen- tration Mean Stand. Mean Domains PeptideCells (μM) counts dev. Factor — No peptide Myoblasts 0 30 175 4687 1.0ELD-CPD CM18-TAT- Myoblasts 5 88 686 19 481 2.9 Cys ELD-CPD dCM18-Myoblasts 2.5 64 864 1264 2.1 dimer TAT-Cys ELD-CPD CM18- Myoblasts 5 65636 3288 2.2 Penetratin- Cys ELD-CPD dCM18- Myoblasts 2.5 71 547 10 9752.4 dimer Penetratin- Cys

The results in Table 2.11 suggest that shuttle peptide dimers are ableto facilitate calcein endosomal escape levels that are comparable to thecorresponding monomers in primary myoblasts.

Example 3 Peptide Shuttle Agents Increase GFP Transduction Efficiency3.1 Protein Transduction Assay

One day before the transduction assay was performed, cells inexponential growth phase were harvested and plated in a 96-well plate(20,000 cells per well). The cells were incubated overnight inappropriate growth media containing FBS (see Example 1). The next day,in separate sterile 1.5 mL tubes, cargo protein at the indicatedconcentration was pre-mixed (pre-incubated) for 1 or 10 min (dependingon the protocol) at 37° C. with the peptide(s) to be tested shuttleagents (0.5 to 5 μM) in 50 μL of fresh medium without serum (unlessotherwise specified). The media in wells was removed and the cells werewashed three times with freshly prepared phosphate buffered saline (PBS)previously warmed at 37° C. The cells were incubated with the cargoprotein/shuttle agent mixture at 37° C. for the indicated time (e.g., 1,5 or 60 min). After the incubation, the cells were quickly washed threetimes with freshly prepared PBS and/or heparin (0.5 mg/mL) previouslywarmed at 37° C. The washes with heparin were required for human THP-1blood cells to avoid undesired cell membrane-bound protein background insubsequent analyses (microscopy and flow cytometry). The cells werefinally incubated in 50 μL of fresh medium with serum at 37° C. beforeanalysis.

3.1a Protocol A: Protein Transduction Assay for Adherent Cells

One day before the transduction assay was performed, cells inexponential growth phase were harvested and plated in a 96-well plate(20,000 cells per well). The cells were incubated overnight inappropriate growth media containing serum (see Example 1). The next day,in separate sterile 1.5-mL tubes, peptides were diluted in steriledistilled water at room temperature (if the cargo is or comprised anucleic acid, nuclease-free water was used). Cargo protein(s) were thenadded to the peptides and, if necessary, sterile PBS or cell culturemedium (serum-free) was added to obtain the desired concentrations ofshuttle agent and cargo in a sufficient final volume to cover the cells(e.g., 10 to 100 μL per well for a 96-well plate). The peptides/cargomixture was then immediately used for experiments. At least threecontrols were included for each experiment, including: (1) peptidesalone (e.g., at highest concentration tested); (2) cargo alone; and (3)without any cargo or shuttle agent. The media in wells was removed,cells were washed once with PBS previously warmed at 37° C., and thecells were incubated with the cargo protein/peptide mixture at 37° C.for the desired length of time. The peptide/cargo mixture in wells wasremoved, the cells were washed once with PBS, and fresh complete mediumwas added. Before analysis, the cells were washed once with PBS one lasttime and fresh complete medium was added.

3.1b Protocol B: Protein Transduction Assay for Suspension Cells

One day before the transduction assay was performed, suspension cells inexponential growth phase were harvested and plated in a 96-well plate(20,000 cells per well). The cells were incubated overnight inappropriate growth media containing serum (see Example 1). The next day,in separate sterile 1.5-mL tubes, peptides were diluted in steriledistilled water at room temperature (if the cargo is or comprised anucleic acid, nuclease-free water was used). Cargo protein(s) were thenadded to the peptides and, if necessary, sterile PBS or cell culturemedium (serum-free) was added to obtain the desired concentrations ofshuttle agent and cargo in a sufficient final volume to resuspend thecells (e.g., 10 to 100 μL per well in a 96-well plate). The shuttleagent/peptide was then immediately used for experiments. At least threecontrols were included for each experiment, including: (1) peptide alone(e.g., at highest concentration tested); (2) cargo alone; and (3)without any cargo or shuttle agent. The cells were centrifuged for 2minutes at 400 g, the medium was then removed and the cells wereresuspended in PBS previously warmed at 37° C. The cells werecentrifuged again 2 minutes at 400 g, the PBS removed, and the cellswere resuspended with the cargo protein/peptide mixture at 37° C. forthe desired length of time. After that, 200 μL of complete medium wasadded directly on the cells. Cells were centrifuged for 2 minutes at 400g and the medium was removed. The pellet was resuspended and washed in200 μL of PBS previously warmed at 37° C. After another centrifugation,the PBS was removed and the cells were resuspended in 50 μL oftrypsin-EDTA solution for 2 min. 200 of complete medium was directlyadded and cells were centrifuged for 2 minutes at 400 g. The medium wasremoved and the cells were resuspended in 200 μL of complete medium.

3.2 Fluorescence Microscopy Analysis

The delivery of fluorescent protein cargo in cytosolic and nuclear cellcompartments was observed with an Olympus IX70™ microscope (Japan)equipped with a fluorescence lamp (Model U-LH100HGAPO) and differentfilters. The Olympus filter U-MF2™ (C54942-Exc495/Em510) was used toobserve GFP and FITC-labeled antibody fluorescent signals. The Olympusfilter HQ-TR™ (V-N41004-Exc555-60/Em645-75) was used to observe mCherry™and GFP antibody fluorescent signals. The Olympus filter U-MWU2™(Exc330/Em385) was used to observe DAPI or Blue Hoechst fluorescentsignals. The cells incubated in 50 μL of fresh medium were directlyobserved by microscopy (Bright-field and fluorescence) at differentpower fields (4× to 40×). The cells were observed using a CoolSNAP-PRO™camera (Series A02D874021) and images were acquired using theImage-Proplus™ software.

3.2a Cell Immuno-Labelling

Adherent cells were plated on a sterile glass strip at 1.5×10⁵ cells perwell in a 24-plate well and incubated overnight at 37° C. For fixation,cells were incubated in 500 μL per well of formaldehyde (3.7% v/v) for15 minutes at room temperature, and washed 3 times for 5 minutes withPBS. For permeabilization, cells were incubated in 500 μL per well ofTriton™ X-100 (0.2%) for 10 minutes at room temperature, and washed 3times for 5 minutes with PBS. For blocking, cells were incubated in 500μL per well of PBS containing 1% BSA (PBS/BSA) for 60 minutes at roomtemperature. Primary mouse monoclonal antibody was diluted PBS/BSA (1%).Cells were incubated in 30 μL of primary antibody overnight at 4° C.Cells were washed 3 times for 5 minutes with PBS. Secondary antibody wasdiluted in PBS/BSA (1%) and cells were incubated in 250 μL of secondaryantibody 30 minutes at room temperature in the dark. Cells were washed 3times for 5 minutes with PBS. Glass strips containing the cells weremounted on microscope glass slides with 10 μL of the mounting mediumFluoroshield™ with DAPI.

3.3 Flow Cytometry Analysis:

The fluorescence of GFP was quantified using flow cytometry (Accuri C6,Becton, Dickinson and Company (BD)). Untreated cells were used toestablish a baseline in order to quantify the increased fluorescence dueto the internalization of the fluorescent protein in treated cells. Thepercentage of cells with a fluorescence signal above the maximumfluorescence of untreated cells, “mean %” or “Pos cells (%)”, is used toidentify positive fluorescent cells. “Relative fluorescence intensity(FL1-A)” corresponds to the mean of all fluorescence intensities fromeach cell with a fluorescent signal after fluorescent protein deliverywith the shuttle agent. Also, the events scanned by flow cytometrycorresponding to cells (size and granularity) were analyzed. Thecellular toxicity (% cell viability) was monitored comparing thepercentage of cells in the total events scanned of treated cellscomparatively to untreated cells.

3.3a Viability Analysis

Where indicated, the viability of cells was assessed with a resazurintest. Resazurin is a sodium salt colorant that is converted from blue topink by mitochondrial enzymes in metabolically active cells. Thiscolorimetric conversion, which only occurs in viable cells, can bemeasured by spectroscopy analysis in order to quantify the percentage ofviable cells. The stock solution of resazurin was prepared in water at 1mg/100 mL and stored at 4° C. 25 μL of the stock solution was added toeach well of a 96-well plate, and cells were incubated at 37° C. for onehour before spectrometry analysis. The incubation time used for theresazurin enzymatic reaction depended on the quantity of cells and thevolume of medium used in the wells.

3.4 Construction and Amino Acid Sequence of GFP

The GFP-encoding gene was cloned in a T5 bacterial expression vector toexpress a GFP protein containing a 6× histidine tag and a serine/glycinerich linker in the N-terminal end, and a serine/glycine rich linker anda stop codon (−) at the C-terminal end. Recombinant GFP protein waspurified as described in Example 1.4. The sequence of the GFP constructwas:

[SEQ ID NO: 60] MHHHHHHGGGGSGGGGSGGASTGTGIR MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAA GITLGMDELYKGGSGGGSGGGSGWIRASSGGREIS- (MW = 31.46 kDa; pl = 6.19) Serine/glycine rich linkers are in bold  GFP sequence is underlined 

3.5 GFP Transduction by CM18-TAT-Cys in HeLa Cells: FluorescenceMicroscopy

HeLa cells were cultured and tested in the protein transduction assaydescribed in Example 3.1. Briefly, GFP recombinant protein wasco-incubated with 0, 3 or 5 μM of CM18-TAT, and then exposed to HeLacells for 1 hour. The cells were observed by bright field andfluorescence microscopy as described in Example 3.2. The resultspresented in FIG. 5 show that GFP was delivered intracellularly to HeLacells in the presence of the shuttle agent CM18-TAT.

3.6 GFP Transduction by Shuttle Agents in HeLa Cells: Dose Responses(CM18-TAT-Cys, dCM18-TAT-Cys, GFP) and Cell Viability

HeLa cells were cultured and tested in the protein transduction assaydescribed in Examples 3.1-3.3. Briefly, GFP recombinant protein wasco-incubated with different concentrations of CM18-TAT-Cys or dimerizedCM18-TAT-Cys (dCM18-TAT-Cys), and then exposed to HeLa cells for 1 hour.The results are shown in Table 3.1 and FIGS. 6A-6B.

TABLE 3.1 Dose response (CM18-TAT) and cell viability, data from FIGS.6A and 6B FIG. 6B Cell FIG. 6A viability Concen- Mean (%) tration (%)Standard (±St. Dev.; Shuttle Cells (μM) (n = 3) deviation n = 3)CM18-TAT-Cys HeLa 0 0.69 0.12 95 ± 4 HeLa 0.5 8.67 0.96 88.4 ± 6   HeLa1 20.03 2.55 90 ± 6 HeLa 3 31.06 5.28 91 ± 5 HeLa 5 36.91 4.33 90 ± 7

Table 3.1 and FIG. 6A show the results of flow cytometry analysis of thefluorescence intensity of HeLa cells transduced with GFP (5 μM) withoutor with 5, 3, 1, and 0.5 μM of CM18-TAT-Cys. Corresponding cellulartoxicity data are presented in Table 3.1 and in FIG. 6B. These resultssuggest that the shuttle agent CM18-TAT-Cys increases the transductionefficiency of GFP in a dose-dependent manner.

TABLE 3.2 Dose response (GFP), data from FIGS. 7A and 7B Conc. of Conc.shuttle of Mean agent GFP (%) Standard Shuttle Cells (μM) (μM) (n = 3)deviation Control HeLa 0 10 0.93 0.08 CM18-TAT-Cys HeLa 5 10 37.1 4.29HeLa 5 5 21.1 2.19 HeLa 5 1 8.56 1.91 Control HeLa 0 10 0.91 0.09dCM18-TAT-Cys HeLa 2.5 10 34.2 3.42 HeLa 2.5 5 22.2 3.17 HeLa 2.5 1 9.382.11

Table 3.2 and FIG. 7 show the results of flow cytometry analysis of thefluorescence intensity of HeLa cells transduced with differentconcentrations of GFP (1 to 10 μM) without or with 5 μM of CM18-TAT-Cys(FIG. 7A) or 2.5 μM dCM18-TAT-Cys (FIG. 7B).

3.7 GFP Transduction in HeLa Cells: Dose Responses of CM18-TAT-Cys andCM18-Penetratin-Cys, and Dimers Thereof

HeLa cells were cultured and tested in the protein transduction assaydescribed in Example 3.1. Briefly, GFP recombinant protein (5 μM) wasco-incubated with different concentrations and combinations ofCM18-TAT-Cys, CM18-Penetratin-Cys, and dimers of each (dCM18-TAT-Cys,dCM18-Penetratin-Cys), and then exposed to HeLa cells for 1 hour. Thecells were subjected to flow cytometry analysis as described in Example3.3. The results are shown in Table 3.3 and FIG. 8, as well as in Table3.4 and FIG. 9.

TABLE 3.3 Data in FIG. 8 Concentration Mean (%) Standard No. in FIG. 8Shuttle agent Cells (μM) (n = 3) deviation Control (“ctrl”) No shuttleHeLa 0 0.43 0.08 1 CM18-TAT-Cys HeLa 0.5 8.75 0.63 2 dCM18-TAT-Cys HeLa0.5 8.86 1.03 3 CM18-Penetratin-Cys HeLa 3 0.59 0.11 4dCM18-Penetratin-Cys HeLa 3 0.73 0.08 1 + 3 CM18-TAT-Cys + HeLa 0.519.52 2.18 CM18Penetratin-Cys 3 2 + 3 dCM18-TAT-Cys + HeLa 0.5 22.443.29 CM18-Penetratin-Cys 3 1 + 4 CM18-TAT-Cys + HeLa 0.5 18.73 1.55dCM18-Penetratin-Cys 3 2 + 4 dCM18-TAT-Cys + HeLa 0.5 17.19 1.93dCM18-Penetratin-Cys 3

The results in Table 3.3 and FIG. 8 show that the transductionefficiency of GFP is increased in HeLa cells using the shuttle agentsCM18-TAT-Cys and dCM18-TAT-Cys (see bars “1” and “2” in FIG. 8).Although no GFP intracellular delivery was observed usingCM18-Penetratin-Cys or dCM18-Penetratin-Cys alone (see bars “3” or “4”in FIG. 8), combination of CM18-TAT-Cys with CM18-Penetratin-Cys(monomer or dimer) improved GFP protein delivery (see four right-mostbars in FIG. 8).

TABLE 3.4 Data in FIG. 9 Concentration Mean (%) Standard No. in FIG. 9Shuttle Cells (μM) (n = 3) deviation Control (“ctrl”) No shuttle HeLa 00.51 0.07 1 CM18-TAT-Cys HeLa 1 20.19 2.19 2 dCM18-TAT-Cys HeLa 1 18.431.89 3 CM18-Penetratin-Cys HeLa 3 0.81 0.07 4 dCM18-Penetratin-Cys HeLa3 0.92 0.08 1 + 3 CM18-TAT-Cys + HeLa 1 30.19 3.44 CM18-Penetratin-Cys 32 + 3 dCM18-TAT-Cys + 1 CM18-Penetratin-Cys HeLa 3 22.36 2.46 1 + 4CM18-TAT-Cys + HeLa 1 26.47 2.25 dCM18-Penetratin-Cys 3 2 + 4dCM18-TAT-Cys + HeLa 1 21.44 3.11 dCM18-Penetratin-Cys 3

The results in Table 3.4 and FIG. 9 show that the transductionefficiency of GFP is increased in HeLa cells using the shuttle agentsCM18-TAT-Cys and dCM18-TAT-Cys (see bars “1” and “2” in FIG. 9).Although no GFP intracellular delivery was observed usingCM18-Penetratin-Cys or dCM18-Penetratin-Cys alone (see bars “3” or “4”in FIG. 9), combination of CM18-TAT-Cys with CM18-Penetratin-Cys(monomer or dimer) improved GFP protein delivery (see four right-mostbars in FIG. 9).

3.8 GFP Transduction by Shuttle Agents in HeLa Cells: Controls

HeLa cells were cultured and tested in the protein transduction assaydescribed in Example 3.1. Briefly, GFP recombinant protein (5 μM) wasco-incubated with 5 μM of each of the following peptide(s): TAT-Cys;CM18; Penetratin-Cys; TAT-Cys+CM18; Penetratin-Cys+CM18; andCM18-TAT-Cys, and then exposed to HeLa cells for 1 hour. GFPfluorescence was visualized by bright field and fluorescence microscopy.The microscopy results (data not shown) showed that GFP was successfullydelivered intracellularly using CM18-TAT-Cys. However, GFP was notsuccessfully delivered intracellularly using single-domain peptides usedalone (CM18, TAT-Cys, Penetratin-Cys) or together (CM18+TAT-Cys,CM18+Penetratin-Cys). These results are consistent with those presentedin Tables 2.1 and 2.2 with respect to the calcein endosome escapeassays.

Example 4 Peptide Shuttle Agents Increase TAT-GFP TransductionEfficiency

The experiments in Example 3 showed the ability of shuttle agents todeliver GFP intracellularly. The experiments presented in this exampleshow that the shuttle agents can also increase the intracellulardelivery of a GFP cargo protein that is fused to a CPD (TAT-GFP).

4.1 Construction and Amino Acid Sequence of TAT-GFP

Construction was performed as described in Example 3.4, except that aTAT sequence was cloned between the 6× histidine tag and the GFPsequences. The 6× histidine tag, TAT, GFP and a stop codon (−) areseparated by serine/glycine rich linkers. The recombinant TAT-GFPprotein was purified as described in Example 1.4. The sequence of theTAT-GFP construct was:

SEQ ID NO: 61] MHHHHHHGGGGSGGGGSGGASTGT GRKKRRQRRRPPQ GGGGSGGGGSGGGTGIRMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGGGSGGGSGWI RASSGGREIS-(MW = 34.06 kDa; pl = 8.36) TAT sequence is underlinedSerine/glycine rich linkers are in bold

4.2 TAT-GFP Transduction by CM18-TAT-Cys in HeLa Cells: Visualisation byFluorescence Microscopy

HeLa cells were cultured and tested in the protein transduction assaydescribed in Example 3.1. Briefly, TAT-GFP recombinant protein (5 μM)was co-incubated with 3 μM of CM18-TAT-Cys and then exposed to HeLacells for 1 hour. Cells and GFP fluorescence were visualized by brightfield and fluorescence microscopy (as described in Example 3.2) at 10×and 40× magnifications, and sample results are shown in FIG. 10. Themicroscopy results revealed that in the absence of CM18-TAT-Cys, TAT-GFPshows a low intensity, endosomal distribution as reported in theliterature. In contrast, TAT-GFP is delivered to the cytoplasm and tothe nucleus in the presence of the shuttle agent CM18-TAT-Cys. Withoutbeing bound by theory, the TAT peptide itself may act as a nuclearlocalization signal (NLS), explaining the nuclear localization ofTAT-GFP. These results show that CM18-TAT-Cys is able to increaseTAT-GFP transduction efficiency and allow endosomally-trapped TAT-GFP togain access to the cytoplasmic and nuclear compartments.

4.3 TAT-GFP Transduction by CM18-TAT-Cys in HeLa Cells: Dose Responsesand Viability of Cells Transduced

HeLa cells were cultured and tested in the protein transduction assaydescribed in Example 3.1. Briefly, TAT-GFP recombinant protein (5 μM)was co-incubated with different concentrations of CM18-TAT-Cys (0, 0.5,1, 3, or 5 μM) and then exposed to HeLa cells for 1 hour. The cells weresubjected to flow cytometry analysis as described in Example 3.3.Results are shown in Table 4.3 and FIG. 11A. Corresponding cellulartoxicity data are presented in FIG. 11B.

TABLE 4.3 Data from FIGS. 11A and 11B FIG. 11B Cell FIG. 11A viabilityConcen- Mean (%) tration (%) Standard (±St. Dev.; Shuttle agent Cells(μM) (n = 3) deviation n = 3) CM18-TAT-Cys HeLa 0 11.79¹ 1.16 100 HeLa0.5 10.19 1.94 84.36 ± 5   HeLa 1 14.46 2.59 89.26 ± 5.26 HeLa 3 28.123.27 93.18 ± 6.28 HeLa 5 35.5² 3.59 95.14 ± 5.28 ¹The fluorescence wasmostly endosomal, as confirmed by fluorescence microscopy. ²Fluorescencewas more diffuse and also nuclear, as confirmed by fluorescencemicroscopy.

Example 5 Peptide Shuttle Agents Increase GFP-NLS TransductionEfficiency and Nuclear Localization

The experiments in Examples 3 and 4 showed the ability of shuttle agentsto deliver GFP and TAT-GFP intracellularly. The experiments presented inthis example show that the shuttle agents can facilitate nucleardelivery of a GFP protein cargo fused to a nuclear localization signal(NLS).

5.1 Construction and Amino Acid Sequence of GFP-NLS

Construction was performed as described in Example 3.4, except that anoptimized NLS sequence was cloned between the GFP sequence and the stopcodon (−). The NLS sequence is separated from the GFP sequence and thestop codon by two serine/glycine rich linkers. The recombinant GFP-NLSprotein was purified as described in Example 1.4. The sequence of theGFP-NLS construct was:

[SEQ ID NO: 62] MHHHHHHGGGGSGGGGSGGASTGIRMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIKVNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYKGGSGGGSGGGSGWIRA SSGGRSSDDEATADSQHAAPPKKKRK VGGSGGGSGGGSGGGRGTEIS- (MW = 34.85 kDa; pl = 6.46)NLS sequence is underlined Serine/glycine rich linkers are in bold

5.2 Nuclear Delivery of GFP-NLS by CM18-TAT-Cys in HeLa Cells in 5Minutes: Visualisation by Fluorescence Microscopy

HeLa cells were cultured and tested in the protein transduction assaydescribed in Example 3.1. Briefly, GFP-NLS recombinant protein (5 μM)was co-incubated with 5 μM of CM18-TAT-Cys, and then exposed to HeLacells. GFP fluorescence was visualized by bright field and fluorescencemicroscopy after 5 minutes (as described in Example 3.2) at 10×, 20× and40× magnifications, and sample results are shown in FIG. 12. Themicroscopy results revealed that GFP-NLS is efficiently delivered to thenucleus in the presence of the shuttle agent CM18-TAT-Cys, after only 5minutes of incubation.

5.3 GFP-NLS Transduction by CM18-TAT-Cys in HeLa Cells: Dose Responsesand Viability of Cells Transduced

HeLa cells were cultured and tested in the protein transduction assaydescribed in Example 3.1. GFP-NLS recombinant protein (5 μM) wasco-incubated with 0, 0.5, 1, 3, or 5 μM of CM18-TAT-Cys, and thenexposed to HeLa cells for 1 hour. The cells were subjected to flowcytometry analysis as described in Example 3.3. Results are shown inTable 5.1 and FIG. 13A. Corresponding cellular toxicity data arepresented in FIG. 13B.

TABLE 5.1 Data from FIGS. 13A and 13B FIG. 13B Cell FIG. 13A viabilityConcen- Mean (%) tration (%) Standard (±St Dev.; Shuttle agent Cells(μM) (n = 3) deviation n = 3) CM18-TAT-Cys HeLa 0 0.90 0.12 100 HeLa 0.59.81 1.63 87.6 ± 4   HeLa 1 18.42 2.47 93 ± 8 HeLa 3 28.09 3.24 94 ± 5HeLa 5 32.26 4.79 93 ± 4

These results show that CM18-TAT-Cys is able to increase GFP-NLStransduction efficiency in HeLa cells in a dose-dependent manner.

5.4 GFP-NLS Transduction by CM18-TAT-Cys, CM18-Penetratin-Cys, andDimers Thereof in HeLa Cells

HeLa cells were cultured and tested in the protein transduction assaydescribed in Example 3.1. GFP-NLS recombinant protein (5 μM) wasco-incubated with different concentrations and combinations ofCM18-TAT-Cys, CM18-Penetratin-Cys, and dimers of each (dCM18-TAT-Cys,dCM18-Penetratin-Cys), and then exposed to HeLa cells for 1 hour. Thecells were subjected to flow cytometry analysis as described in Example3.3. The results are shown in Tables 5.2 and 5.3, and in FIGS. 14 and15.

TABLE 5.2 Data in FIG. 14 Concen- Mean No. in tration (%) Standard FIG.14 Shuttle agent Cells (μM) (n = 3) deviation ctrl No shuttle HeLa 00.41 0.10 1 CM18-TAT-Cys HeLa 0.5 7.64 0.85 2 dCM18-TAT-Cys HeLa 0.58.29 0.91 3 CM18-Penetratin- HeLa 3 0.43 0.08 Cys 4 dCM18-Penetratin-HeLa 3 0.85 0.07 Cys 1 + 3 CM18-TAT-Cys + HeLa 0.5 21.1 2.47CM18-Penetratin- 3 Cys 2 + 3 dCM18-TAT-Cys + HeLa 0.5 19.22 2.73CM18-Penetratin- 3 Cys 1 + 4 CM18-TAT-Cys + HeLa 0.5 23.44 2.51dCM18-Penetratin- 3 Cys 2 + 4 dCM18-TAT-Cys + HeLa 0.5 19.47 2.16dCM18-Penetratin- 3 Cys

TABLE 5.3 Data in FIG. 15 Concen- Mean No. in tration (%) Standard FIG.15 Shuttle agent Cells (μM) (n = 3) deviation ctrl No shuttle HeLa 00.44 0.12 1 CM18-TAT-Cys HeLa 1 15.56 2.24 2 dCM18-TAT-Cys HeLa 1 17.832.13 3 CM18-Penetratin- HeLa 3 0.68 0.05 Cys 4 dCM18-Penetratin- HeLa 30.84 0.07 Cys 1 + 3 CM18-TAT-Cys + HeLa 1 27.26 3.61 CM18-Penetratin- 3Cys 2 + 3 dCM18-TAT-Cys + HeLa 1 25.47 3.77 CM18-Penetratin- 3 Cys 1 + 4CM18-TAT-Cys + HeLa 1 31.47 4.59 dCM18-Penetratin- 3 Cys 2 + 4dCM18-TAT-Cys + HeLa 1 28.74 2.93 dCM18-Penetratin- 3 Cys

The results in Tables 5.2 and 5.3 and FIGS. 14 and 15 show that thetransduction efficiency of GFP-NLS is increased in HeLa cells using theshuttle agents CM18-TAT-Cys and dCM18-TAT-Cys (see bars “1” and “2” inFIGS. 14 and 15). Although no GFP-NLS intracellular delivery wasobserved using CM18-Penetratin-Cys or dCM18-Penetratin-Cys alone (seebars “3” and “4” in FIGS. 14 and 15), combination of CM18-TAT-Cys withCM18-Penetratin-Cys (monomer or dimer) improved GFP-NLS intracellulardelivery (see four right-most bars in FIGS. 14 and 15).

5.5 GFP-NLS Transduction by Shuttle Agents in HeLa Cells: 5 Min v. 1 hIncubation; with or without FBS

HeLa cells were cultured and tested in the protein transduction assaydescribed in Example 3.1. GFP-NLS recombinant protein (5 μM) wasco-incubated with either CM18-TAT-Cys (3.5 μM) alone or withdCM18-Penetratin-Cys (1 μM). Cells were incubated for 5 minutes or 1hour in plain DMEM media (“DMEM”) or DMEM media containing 10% FBS(“FBS”), before being subjected to flow cytometry analysis as describedin Example 3.3. The results are shown in Table 5.4, and in FIG. 16.Cells that were not treated with shuttle agent or GFP-NLS (“ctrl”), andcells that were treated with GFP-NLS without shuttle agent (“GFP-NLS 5μM”) were used as controls.

TABLE 5.4 Data in FIG. 16 No. in FIG. Incubation Shuttle Conc. Mean (%)Standard Shuttle 16 Cells Medium time (μM) (n = 3) deviation No shuttle(Ctrl) 1 HeLa DMEM 1 h 0 0.59 0.09 GFP-NLS alone 2 HeLa DMEM 1 h 0 1.190.31 CM18-TAT-Cys 3 HeLa DMEM 1 h 3.5 20.69 1.19 4 HeLa FBS 1 h 3.513.20 0.82 CM18-TAT-Cys 5 HeLa DMEM 5 min 3.5 20.45 4.26 6 HeLa FBS 5min 3.5 10.83 1.25 No shuttle (Ctrl) 1 HeLa DMEM 1 h 0 0.53 0.11 GFP-NLSalone 2 HeLa DMEM 1 h 0 1.25 0.40 CM18-TAT-Cys + 3 HeLa DMEM 1 h 3.5127.90 2.42 dCM18-Penetratin- 4 HeLa FBS 1 h 3.51 8.35 0.46 CysCM18-TAT-Cys + 5 HeLa DMEM 5 min 3.51 24.10 2.76 dCM18-Penetratin- 6HeLa FBS 5 min 3.51 5.02 0.72 Cys

The results in Table 5.4 and FIG. 16 show that the addition of even arelatively low amount of the dimer dCM18-Penetratin-Cys (1 μM;“dCM18pen”) to the CM18-TAT-Cys monomer improved GFP-NLS transductionefficiency. Interestingly, intracellular GFP-NLS delivery was achievedin as little as 5 minutes of incubation, and delivery was stillachievable (although reduced) in the presence of FBS.

5.6 GFP-NLS Transduction by Shuttle Agents in THP-1 Suspension Cells

The ability of the shuttle agents to deliver GFP-NLS intracellularly wastested in THP-1 cells, which is an acute monocytic leukemia cell linethat grows in suspension. THP-1 cells were cultured (see Example 1) andtested in the protein transduction assay described in Example 3.1.GFP-NLS recombinant protein (5 μM) was co-incubated with or without 1 μMCM18-TAT-Cys, and exposed to the THP-1 cells for 5 minutes, before beingsubjected to flow cytometry analysis as described in Example 3.3. Theresults are shown in Table 5.5 and in FIG. 17A. Corresponding cellulartoxicity data are presented in FIG. 17B.

TABLE 5.5 Data in FIGS. 17A and 17B FIG. 17B Cell FIG. 17A viabilityShuttle Mean (%) Conc. (%) Standard (±St Dev.; Shuttle Cells (μM) (n =3) deviation n = 3) No shuttle (Ctrl) THP-1 0 1.23 0.16 95 ± 4 GFP-NLSalone 0 2.49 0.37 96 ± 3 CM18-TAT-Cys 1 38.1 4.16 85 ± 6

The results in Table 5.5 and FIG. 17 demonstrate the ability of theshuttle agents to deliver protein cargo intracellularly to a humanmonocytic cell line grown in suspension.

Example 6 Peptide Shuttle Agents Increase Transduction Efficiency of anFITC-Labeled Anti-Tubulin Antibody

The experiments in Examples 3-5 showed the ability of shuttle agents toincrease the transduction efficiency of GFP, TAT-GFP, and GFP-NLS. Theexperiments presented in this example show that the shuttle agents canalso deliver a larger protein cargo: an FITC-labeled anti-tubulinantibody. The FITC-labeled anti-tubulin antibody was purchased from(Abcam, ab64503) and has an estimated molecular weight of 150 KDa. Thedelivery and microscopy protocols are described in Example 3.

6.1 Transduction of a Functional Antibody by CM18-TAT-Cys in HeLa Cells:Visualization by Microscopy

FITC-labeled anti-tubulin antibody (0.5 μM) was co-incubated with 5 μMof CM18-TAT-Cys and exposed to HeLa cells for 1 hour. Antibody deliverywas visualized by bright field (20×) and fluorescence microscopy (20×and 40×). As shown in FIG. 18, fluorescent tubulin fibers in thecytoplasm were visualized, demonstrating the functionality of theantibody inside the cell.

6.2 Transduction of a Functional Antibody by CM18-TAT-Cys,CM18-Penetratin-Cys, and Dimers in HeLa Cells: Flow Cytometry

HeLa cells were cultured and tested in the protein transduction assaydescribed in Example 3.1. FITC-labeled anti-tubulin antibody (0.5 μM)was co-incubated with 3.5 μM of CM18-TAT-Cys, CM18-Penetratin-Cys ordCM18-Penetratin-Cys, or a combination of 3.5 μM of CM18-TAT-Cys and 0.5μM of dCM18-Penetratin-Cys, and exposed to HeLa cells for 1 hour. Thecells were subjected to flow cytometry analysis as described in Example3.3. Results are shown in Table 6.1 and FIG. 19A. Corresponding cellulartoxicity data are presented in FIG. 19B.

TABLE 6.1 Data from FIG. 19A and 19B Shuttle FIG. 19A FIG. 19B Conc.Mean (%) Standard Cell viability (%) Domains Shuttle agent Cells (μM) (n= 3) deviation (± St. Dev.; n = 3) — No shuttle HeLa 0 0.9 0.06 98 ±1.0  (“Ctrl”) — Antibody alone HeLa 0 2.66 0.61 96 ± 3.4  (“antibody”)ELD-CPD CM18-TAT-Cys HeLa 3.5 36.56 4.06 95 ± 4.06 CM18-Penetratin-CysHeLa 3.5 53.05 9.5 73 ± 9.5  ELD-CPD dimer dCM18-Penetratin- HeLa 3.550.23 9.12 74 ± 9.0  Cys ELD-CPD + CM18-TAT-Cys + HeLa 3.5 47.19 8.5 93± 8.5  ELD-CPD dimer dCM18-Penetratin- 0.5 Cys

The results in Table 6.1 and FIGS. 18 and 19 show that both CM18-TAT-Cysand CM18-Penetratin-Cys facilitate intracellular delivery of anFITC-labeled anti-tubulin antibody. In contrast to the results with GFP,TAT-GFP, and GFP-NLS in Examples 3-5, CM18-Penetratin-Cys was able todeliver the antibody cargo intracellularly when used alone (withoutCM18-TAT-Cys). However, combination of CM18-TAT-Cys anddCM18-Penetratin-Cys allowed for higher intracellular delivery ascompared with CM18-TAT-Cys alone, and with less cell toxicity ascompared to CM18-Penetratin-Cys and dCM18-Penetratin-Cys (see FIGS. 19Aand 19B).

Example 7 CM18-TAT-Cys Enables Intracellular Plasmid DNA Delivery butPoor Plasmid Expression

The ability of the CM18-TAT-Cys shuttle agent to deliver plasmid DNAintracellularly was tested in this example on HEK293A cells using aplasmid encoding GFP.

7.1 Transfection Assay in HEK293A Cells

One day before the transfection assay was performed, mammalian cells(HEK293A) in exponential growth phase were harvested and plated in a24-well plate (50,000 cells per well). The cells were incubatedovernight in appropriate growth media containing FBS. The next day, inseparate sterile 1.5 mL tubes, pEGFP labeled with a Cy5™ fluorochromewas mixed for 10 min at 37° C. with CM18-TAT-Cys (0.05, 0.5, or 5 μM) infresh PBS at a final 100 μL volume. The media in wells was removed andthe cells were quickly washed three times with PBS and 500 μL of warmmedia without FBS was added. The pEGFP and CM18-TAT-Cys solution wasadded to the cells and incubated at 37° C. for 4 hours. After theincubation, cells were washed with PBS and fresh media containing FBSwas added. Cells were incubated at 37° C. before being subjected to flowcytometry analysis as described in Example 3.

7.2 Plasmid DNA Delivery with CM18-TAT-Cys

Plasmid DNA (pEGFP) was labeled with a Cy5™ dye following themanufacturer's instructions (Mirus Bio LLC). Cy5™ Moiety did notinfluence transfection efficiency when compared to unlabelled plasmidusing standard transfection protocol (data not shown). Flow cytometryanalysis allowed quantification of Cy5™ emission, corresponding to DNAintracellular delivery, and GFP emission, corresponding to successfulnuclear delivery, DNA transcription and protein expression. The resultsare shown in Table 7.1 and in FIG. 20.

TABLE 7.1 Data from FIG. 20 GFP Cy5 ™ expression fluorescence Mean Mean(% of cells Cy5 ™ with GFP DNA signal Standard signal; Standard Sample(ng) (n = 3) deviation n = 3) deviation pEGFP-Cy5 500  914 0 0.0% n/aalone CM18-TAT- 500 1450 120 0.0% n/a Cys, 0.05 μM CM18-TAT- Cys, 0.5 μM500 8362 294 0.0% n/a CM18-TAT- 500 140 497   3977 0.1% n/a Cys, 5 μM

The results shown in Table 7.1 and in FIG. 20 show that CM18-TAT-Cys wasable to increase the intracellular delivery the plasmid DNA when used at0.05, 0.5 and 5 μM concentrations, as compared to cell incubated withDNA alone (“pEGFP-Cy5”). However, no expression of GFP was detected inthe cells, which suggests that very little of the plasmid DNA gainedaccess to the cytoplasmic compartment, allowing nuclear localization.Without being bound by theory, it is possible that the plasmid DNA wasmassively sequestered in endosomes, preventing escape to the cytoplasmiccompartment. Salomone et al., 2013 reported the use of a CM18-TAT11hybrid peptide to deliver plasmid DNA intracellularly. They used theluciferase enzyme reporter assay to assess transfection efficiency,which may not be ideal for quantifying the efficiency ofcytoplasmic/nuclear delivery, as the proportion of plasmid DNA that issuccessfully released from endosomes and delivered to the nucleus may beoverestimated due to the potent activity of the luciferase enzyme. Inthis regard, the authors of Salomone et al., 2013 even noted that theexpression of luciferase occurs together with a massive entrapment of(naked) DNA molecules into vesicles, which is consistent with theresults shown in Table 7.1 and in FIG. 20.

7.3 Plasmid DNA Delivery by Peptides in HeLa Cells

Following the poor transfection efficiency of the peptide CM18-TAT-Cys(0.1%, see Table 7.1) observed in HEK293A cells, the experiment wasrepeated with CM18-TAT-Cys in another cell line (HeLa), along with otherpeptides listed in Table 1.3, Table B1, and Table C1.

One day before the transfection assay was performed, HeLa cells inexponential growth phase were harvested and plated in a 96-well plate(10,000 cells per well). The cells were incubated overnight inappropriate growth media containing FBS. The next day, in separatesterile 1.5 mL tubes, the peptide to be tested and the polynucleotidecargo (pEGFP-C1) were mixed for 10 min at 37° C. in serum-free medium ata final volume of 50 μL. The media in wells was removed and the cellswere quickly washed one time with PBS at 37° C. The mix containing thepeptide to be tested and the polynucleotide cargo was added to the cellsand incubated at 37° C. for the indicated period of time (e.g., 1 min, 1h or 4 h). After the incubation, cells were washed one time with PBS at37° C. and fresh media containing FBS was added. Cells were incubated at37° C. before being subjected to flow cytometry analysis as described inExample 3.2, to qualify transfection efficiency (i.e., cells expressionEGFP) and viability. Results are shown in Table 7.2.

TABLE 7.2 DNA transfection in HeLa cells using peptides Mean % Cellcells with viability GFP signal (%) (±St. Dev.; (±St. Dev.; Peptide n =3) n = 3) No peptide (neg. control) 0.0 ± 0.0 100 PTD4-KALA 0.85 ± 0.0453.64 ± 3.91 FSD9 0.81 ± 0.09  36.1 ± 3.41 KALA 0.79 ± 0.06 90.62 ± 4.16His-CM18-Transportan 0.55 ± 0.01 11.89 ± 1.07 FSD12 0.37 ± 0.14  58.6 ±2.07 dCM18-Pen-Cys 0.35 ± 0.02  3.36 ± 0.26 His-CM18-PTD4-His 0.34 ±0.03  29.4 ± 2.38 FSD2 0.34 ± 0.00 55.77 ± 4.19 Pep1-KALA 0.31 ± 0.0392.47 ± 3.42 F5D25 0.31 ± 0.02 98.19 ± 1.19 FSD7 0.29 ± 0.07  60.9 ±7.59 CM18-PTD4-His 0.26 ± 0.01  29.5 ± 0.21 FSD19 0.24 ± 0.00 97.41 ±2.07 FSD10 0.21 ± 0.01 72.36 ± 8.61 FSD24 0.20 ± 0.00 96.45 ± 3.02 FSD150.18 ± 0.02  98.3 ± 1.07 12His-CM18-PTD4 0.18 ± 0.01 97.55 ± 1.57CM18-L1-PTD4 0.16 ± 0.01  84.3 ± 5.64 FSD33 0.15 ± 0.01  75.3 ± 4.19TAT-LAH4 0.15 ± 0.00 96.17 ± 2.70 CM18-L2-PTD4 0.14 ± 0.01  93.7 ± 3.07M-His-CM18-TAT-Cys 0.13 ± 0.01 33.1 ± 0.4 FSD42 0.12 ± 0.02 96.67 ± 1.96FSD11 0.11 ± 0.01  46.2 ± 1.35 Xentry-KALA  0.1 ± 0.02  75.3 ± 4.29 FSD5 0.1 ± 0.01 93.24 ± 8.63 3HA-CM18-PTD4 0.09 ± 0.01 51.48 ± 4.83 FSD320.08 ± 0.02 98.36 ± 0.15 CM18-TAT-Cys 0.08 ± 0.01 96.28 ± 1.86 FSD8 0.06± 0.84  42.3 ± 6.42 CM18-L3-PTD4 0.06 ± 0.01  98.4 ± 0.83 3His-CM18-PTD40.06 ± 0.01 82.05 ± 6.81 CM18-PTD4 0.06 ± 0.01 49.64 ± 5.06 TAT-CM180.06 ± 0.01 44.79 ± 4.17 HA-CM18-PTD4 0.06 ± 0.0  53.21 ± 4.62His-CM18-TAT 0.05 ± 0.01  13.6 ± 0.18 VSVG-PTD4 0.05 ± 0.01 96.21 ± 2.579His-CM18-PTD4 0.04 ± 0.01 98.72 ± 0.93 JST-PTD4 0.04 ± 0.01  70.2 ±5.39 His-CM18-PTD4 0.04 ± 0.01  63.2 ± 4.07 FSD23 0.04 ± 0.00 98.18 ±1.03 FSD20 0.04 ± 0.00 20.49 ± 3.53 FSD38 0.02 ± 0.00   95 ± 2.78 FSD160.02 ± 0.00 99.07 ± 0.73 FSD26 0.02 ± 0.00  97.2 ± 1.53 FSD27 0.02 ±0.00   98 ± 0.63 FSD35 0.02 ± 0.00 96.14 ± 1.67 CM18 0.02 ± 0.00  99.4 ±0.14 FSD30 0.02 ± 0.00 97.41 ± 2.06 His-CM18-9Arg 0.02 ± 00.0 31.63 ±0.11 FSD21 0.01 ± 0.00 96.17 ± 1.69 6His-PTD4 0.01 ± 0.00 97.25 ± 1.34FSD31 0.01 ± 0.00 98.43 ± 0.43 FSD34 0.01 ± 0.00 96.43 ± 2.41 FSD36 0.01± 0.00 97.05 ± 1.99 FSD40 0.01 ± 0.00 98.63 ± 1.08 FSD41 0.01 ± 0.0094.38 ± 2.81 FSD28 0.01 ± 0.00   97 ± 1.11 CM18-Pen-Cys 0.01 ± 0.0  16.1 ± 0.12 PTD4 0.00 ± 0.01  98.2 ± 0.69 FSD39 0.00 ± 0.00  99.2 ±0.61 His-CMH18-PTD4 0.00 ± 0.00 95.15 ± 2.33 Penetratin 0.00 ± 0.0097.42 ± 1.03 C(LLKK)3 0.00 ± 0.00 81.74 ± 2.34

All the peptides tested in Table 7.2 showed transfection efficiencieslower than 1%. Furthermore, the low transfection efficiency ofCM18-TAT-Cys was confirmed in HeLa cells (0.08%). These results showthat peptides which are suitable for delivering polypeptide cargos maynot necessarily be suitable for delivering plasmid DNA. For example, theshuttle agent His-CM18-PTD4-His is shown herein to effectively transducepolypeptide cargos (e.g., see Example 10), yet this peptide displayedonly a DNA plasmid transfection efficiency of 0.34% (Table 7.2).

Example 8 Addition of a Histidine-Rich Domain to Shuttle Agents FurtherImproves GFP-NLS Transduction Efficiency 8.1 GFP-NLS Transduction byHis-CM18-TAT-Cys in HeLa Cells: Visualization by Microscopy

GFP-NLS (5 μM; see Example 5) was co-incubated with 5 μM of CM18-TAT-Cysor His-CM18-TAT and exposed to HeLa cells for 1 hour. Nuclearfluorescence of intracellularly delivered GFP-NLS was confirmed byfluorescence microscopy (data not shown), indicating successful deliveryof GFP-NLS to the nucleus.

8.2 GFP-NLS Transduction by His-CM18-TAT in HeLa Cells: Flow Cytometry

HeLa cells were cultured and tested in the protein transduction assaydescribed in Example 3.1. GFP-NLS (5 μM) was co-incubated with 0, 1, 3or 5 μM of CM18-TAT-Cys or His-CM18-TAT, and exposed to HeLa cells for 1hour. The cells were subjected to flow cytometry analysis as describedin Example 3.3. Results are shown in Table 8.1 and FIG. 21ACorresponding cellular toxicity data are presented in FIG. 21B.

TABLE 8.1 Data from FIGS. 21A and 21B FIG. 21B FIG. 21A Cell Mean (%)viability Shuttle cell with (%) Conc. GFP signal Standard (±St. Dev.;Shuttle agent Cells (μM) (n = 3) deviation n = 3) Ctrl HeLa 0 0.63 0.1096 ± 3.17 (no shuttle, no GFP-NLS) GFP-NLS 0 0.93 0.26 97 ± 2.05 aloneCM18-TAT- 5 20.54 3.51 81 ± 6.34 Cys 3 15.66 2.18 89 ± 5.37 1 8.64 1.1194 ± 4.28 Ctrl HeLa 0 0.51 0.28 95 ± 4.19 (no shuttle, no GFP-NLS)GFP-NLS 0 1.07 0.42 96 ± 3.16 alone His-CM18- 5 41.38 4.59 86 ± 4.59 TAT3 29.58 3.61 91 ± 5.18 1 8.45 1.83 95 ± 3.05

Strikingly, the results in Table 8.1 and in FIG. 21 show thatHis-CM18-TAT was able to increase GFP-NLS protein transductionefficiency by about 2-fold at 3 μM and 5 μM concentrations, as comparedto CM18-TAT-Cys. These results suggest that adding a histidine-richdomain to a shuttle agent comprising an ELD and CPD, may significantlyincrease its polypeptide cargo transduction efficiency. Alternatively orin parallel, combining the shuttle agents with a further independentsynthetic peptide containing a histidine-rich domain fused to a CPD (butlacking an ELD) may provide a similar advantage for proteintransduction, with the added advantage of allowing the concentration ofthe histidine-rich domain to be varied or controlled independently fromthe concentration of the shuttle agent Without being bound by theory,the histidine-rich domain may act as a proton sponge in the endosome,providing another mechanism of endosomal membrane destabilization.

Example 9 His-CM18-PTD4 Increases Transduction Efficiency and NuclearDelivery of GFP-NLS, mCherry®-NLS and FITC-Labeled Anti-Tubulin Antibody9.1 Protein Transduction Protocols Protocol A: Protein TransductionAssay for Delivery in Cell Culture Medium

One day before the transduction assay was performed, cells inexponential growth phase were harvested and plated in a 96-well plate(20,000 cells per well). The cells were incubated overnight inappropriate growth media containing FBS (see Example 1). The next day,in separate sterile 1.5-mL tubes, cargo protein at the desiredconcentration was pre-mixed (pre-incubated) for 10 min at 37° C. withthe desired concentration of shuttle agents in 50 μL of fresh serum-freemedium (unless otherwise specified). The media in wells was removed andthe cells were washed one to three times (depending on the type of cellsused) with PBS previously warmed at 37° C. The cells were incubated withthe cargo protein/shuttle agent mixture at 37° C. for the desired lengthof time. After the incubation, the cells were washed three times withPBS and/or heparin (0.5 mg/mL) previously warmed at 37° C. The washeswith heparin were used for human THP-1 blood cells to avoid undesiredcell membrane-bound protein background in subsequent analyses(microscopy and flow cytometry). The cells were finally incubated in 50μL of fresh medium with serum at 37° C. before analysis.

Protocol B: Protein Transduction Assay for Adherent Cells in PBS

One day before the transduction assay was performed, cells inexponential growth phase were harvested and plated in a 96-well plate(20,000 cells per well). The cells were incubated overnight inappropriate growth media containing serum (see Example 1). The next day,in separate sterile 1.5-mL tubes, shuttle agents were diluted in steriledistilled water at room temperature (if the cargo is or comprised anucleic acid, nuclease-free water was used). Cargo protein(s) were thenadded to the shuttle agents and, if necessary, sterile PBS was added toobtain the desired concentrations of shuttle agent and cargo in asufficient final volume to cover the cells (e.g., 10 to 100 μL per wellfor a 96-well plate). The shuttle agent/cargo mixture was thenimmediately used for experiments. At least three controls were includedfor each experiment, including: (1) shuttle agent alone (e.g., athighest concentration tested); (2) cargo alone; and (3) without anycargo or shuttle agent. The media in wells was removed, cells werewashed once with PBS previously warmed at 37° C., and the shuttleagent/cargo mixture was then added to cover all cells for the desiredlength of time. The shuttle agent/cargo mixture in wells was removed,the cells were washed once with PBS, and fresh complete medium wasadded. Before analysis, the cells were washed once with PBS and freshcomplete medium was added.

Protocol C: Protein Transduction Assay for Suspension Cells in PBS

One day before the transduction assay was performed, suspension cells inexponential growth phase were harvested and plated in a 96-well plate(20,000 cells per well). The cells were incubated overnight inappropriate growth media containing serum (see Example 1). The next day,in separate sterile 1.5-mL tubes, shuttle agents were diluted in steriledistilled water at room temperature (if the cargo is or comprised anucleic acid, nuclease-free water was used). Cargo protein(s) were thenadded to the shuttle agents and, if necessary, sterile PBS or cellculture medium (serum-free) was added to obtain the desiredconcentrations of shuttle agent and cargo in a sufficient final volumeto resuspend the cells (e.g., 10 to 100 μL per well in a 96-well plate).The shuttle agent/cargo mixture was then immediately used forexperiments. At least three controls were included for each experiment,including: (1) shuttle agent alone (e.g., at highest concentrationtested); (2) cargo alone; and (3) without any cargo or shuttle agent.The cells were centrifuged for 2 minutes at 400 g, the medium was thenremoved and the cells were resuspended in PBS previously warmed at 37°C. The cells were centrifuged again 2 minutes at 400 g, the PBS removed,and the cells were resuspended in the shuttle agent/cargo mixture. Afterthe desired incubation time, 100 μL of complete medium was addeddirectly on the cells. Cells were centrifuged for 2 minutes at 400 g andthe medium was removed. The pellet was resuspended and washed in 200 μLof PBS previously warmed at 37° C. After another centrifugation, the PBSwas removed and the cells were resuspended in 100 μL of complete medium.The last two steps were repeated one time before analysis.

9.2 GFP-NLS Transduction by His-CM18-PTD4 in HeLa Cells Using Protocol Aor B: Flow Cytometry

To compare the effects of different protocols on shuttle agenttransduction efficiency, HeLa cells were cultured and tested in theprotein transduction assays using Protocol A or B as described inExample 9.1. Briefly, GFP-NLS recombinant protein (5 μM; see Example5.1) was co-incubated with 10 μM of His-CM18-PTD4 and exposed to HeLacells for 1 hour using Protocol A, or was co-incubated with 35 μM ofHis-CM18-PTD4 and exposed to HeLa cells for 10 seconds using Protocol B.The cells were subjected to flow cytometry analysis as described inExample 3.3. Results are shown in Table 9.1 and FIG. 22A. (“Pos cells(%)” is the percentage of cells emanating a GFP signal).

TABLE 9.1 Comparison of Protein Transduction Protocols A and B: Datafrom FIG. 22A Conc. Mean % Conc. of cells with Cell of GFP- GFP signalviability Pro- shuttle NLS (± St. Dev.; (%) (± St. tocol Shuttle Cells(μM) (μM) n = 3) Dev.; n = 3) B None (“Ctrl”) HeLa 0 5 0.53 ± 0.07 100 AHis-CM18- HeLa 10 5 25.4 ± 3.6  96.4 ± 2.7 PTD4 B His-CM18- HeLa 35 578.3 ± 5.3  94.6 ± 0.4 PTD4

The above results show that higher protein transduction efficiency forthe cargo GFP-NLS using the shuttle agent His-CM18-PTD4 was obtainedusing Protocol B, as compared to Protocol A.

9.3 GFP-NLS Transduction by His-CM18-PTD4 in HeLa Cells Using ProtocolB: Flow Cytometry

A dose response experiment was performed to evaluate the effect ofHis-CM18-PTD4 concentration on protein transduction efficiency. HeLacells were cultured and tested in the protein transduction assaydescribed in Protocol B of Example 9.1. Briefly, GFP-NLS recombinantprotein (5 μM; see Example 5.1) was co-incubated with 0, 50, 35, 25, or10 μM of His-CM18-PTD4, and then exposed to HeLa cells for 10 seconds.The cells were subjected to flow cytometry analysis as described inExample 3.3. Results are shown in Table 9.2 and FIG. 228.

TABLE 9.2 Dose response of shuttle agent using Protocol B: Data fromFIG. 22B Conc. Mean % Cell Conc. of cells with viability of GFP- GFPsignal (%) Pro- shuttle NLS (± St. Dev.; (± St. Dev.; tocol ShuttleCells (μM) (μM) n = 3) n = 3) B None (“Ctrl”) HeLa 0 5 0.13 ± 0.1 100 ±0   His-CM18- 50 5 73.2 ± 5.2 69.2 ± 2.7 PTD4 35 5 77.7 ± 7.8 79.6 ± 5.925 5 62.1 ± 6.1 95.3 ± 3.7 10 5 25.3 ± 3.6 96.3 ± 2.3

The above results show that His-CM18-PTD4 is able to increase GFP-NLStransduction efficiency in HeLa cells in a dose-dependent manner.

9.4 GFP-NLS Transduction by His-CM18-PTD4 in HeLa Cells Using ProtocolB: Visualization by Microscopy

GFP-NLS recombinant protein (5 μM; see Example 5.1) was co-incubatedwith 35 μM of His-CM18-PTD4, and then exposed to HeLa cells for 10seconds using Protocol B as described in Example 9.1. The cells werethen subjected to fluorescence microscopy analysis as described inExamples 3.2 and 3.2a.

For the sample results shown in FIGS. 23 and 24, GFP fluorescence of theHeLa cells was immediately visualized by bright field and fluorescencemicroscopy at 4×, 20× and 40× magnifications after the final washingstep.

In FIG. 23, the upper panels in FIGS. 23A, 23B and 23C show nucleilabelling (DAPI) at 4×, 20× and 40× magnifications, respectively, whilethe lower panels show corresponding GFP-NLS fluorescence. In FIG. 23C,white triangle windows indicate examples of areas of co-labellingbetween nuclei (DAPI) and GFP-NLS signals. In FIG. 23D, the upper andbottom panels show sample bright field images of the HeLa cells, and themiddle panel shows the results of a corresponding FACS analysis(performed as described in Example 3.3), which indicates the percentageof cells in a 96-plate with a GFP signal. No significant GFPfluorescence was observed in negative control samples (i.e., cellsexposed to GFP-NLS without any shuttle agent data not shown).

FIG. 24 shows bright field (FIG. 24A) and fluorescent images (FIG. 24B).The inset in FIG. 24B shows the results of a corresponding FACS analysis(performed as described in Example 3.3), which indicates the percentageof cells in a 96-plate well with a GFP signal. No significant GFPfluorescence was observed in negative control samples (i.e., cellsexposed to GFP-NLS without any shuttle agent; data not shown).

For the sample results shown in FIG. 25, the HeLa cells were fixed,permeabilized and subjected to immuno-labelling as described in Example3.2a before visualization by fluorescence microscopy as described inExample 3.2. GFP-NLS was labelled using a primary mouse monoclonalanti-GFP antibody (Feldan, # A017) and a secondary goat anti-mouseAlexa™-594 antibody (Abcam #150116). The upper panels in FIGS. 25A and25B show nuclei labelling (DAPI), and the lower panels showcorresponding labelling for GFP-NLS. FIGS. 25A and 25B show sampleimages at 20× and 40× magnifications, respectively. White trianglewindows indicate examples of areas of co-labelling between nuclei andGFP-NLS. No significant GFP-NLS labelling was observed in negativecontrol samples (i.e., cells exposed to GFP-NLS without any shuttleagent; data not shown).

FIG. 26 shows sample images captured with confocal microscopy at 63×magnification of living cells. FIG. 26A shows a bright field image,while FIG. 26B shows the corresponding fluorescent GFP-NLS. FIG. 26C isan overlay between the images in FIG. 26A and FIG. 26B. No significantGFP-NLS fluorescence was observed in negative control samples (i.e.,cells exposed to GFP-NLS without any shuttle agent data not shown).

9.4a FTIC-Labeled Anti-Tubulin Antibody Transduction by His-CM18-PTD4 inHeLa Cells Using Protocol B: Visualization by Microscopy

FITC-labeled anti-tubulin antibody (0.5 μM; Abcam, ab64503) wasco-incubated with 50 μM of His-CM18-PTD4, and then exposed to HeLa cellsfor 10 seconds using Protocol B as described in Example 9.1. The cellswere then subjected to fluorescence microscopy analysis as described inExamples 3.2 and 3.2a, wherein the FITC fluorescence of the anti-tubulinantibody in the HeLa cells was immediately visualized by bright fieldand fluorescence microscopy at 20× magnification after the final washingstep. Sample results are shown in FIGS. 24C and 24D. No significant FITCfluorescence was observed in negative control samples (i.e., cellsexposed to the FITC-labeled anti-tubulin antibody without any shuttleagent; data not shown).

Overall, the results in Examples 9.4 and 9.4a show that GFP-NLS andFITC-labeled anti-tubulin antibody cargos are successfully transducedand delivered to the nucleus and/or the cytosol of HeLa cells in thepresence of the shuttle agent His-CM18-PTD4.

9.5 GFP-NLS Kinetic Transduction by His-CM18-PTD4 in HeLa Cells:Visualization by Microscopy

GFP-NLS recombinant protein (5 μM; see Example 5.1) was co-incubatedwith 50 μM of His-CM18-PTD4, and then exposed to HeLa cells for 10seconds using Protocol B as described in Example 9.1. After a washingstep, the GFP fluorescence of the HeLa cells was immediately visualizedby fluorescence microscopy (Example 3.2) at 20× magnification afterdifferent intervals of time. Typical results are shown in FIG. 27, inwhich fluorescence microscopy images were captured after 45, 75, 100,and 120 seconds (see FIGS. 27A, 27B, 27C and 27D, respectively).

As shown in FIG. 27A, diffuse cellular GFP fluorescence was generallyobserved after 45 seconds, with areas of lower GFP fluorescence in thenucleus in many cells. These results suggest predominantly cytoplasmicand low nuclear distribution of the GPF-NLS delivered intracellularlyvia the shuttle agent after 45 seconds. FIGS. 27B-27D show the gradualredistribution of GFP fluorescence to the cell nuclei at 75 seconds(FIG. 27B), 100 seconds (FIG. 27 C), and 120 seconds (FIG. 27D)following exposure to the His-CM18-PTD4 shuttle agent and GFP-NLS cargo.No significant cellular GFP fluorescence was observed in negativecontrol samples (i.e., cells exposed to GFP-NLS without any shuttleagent; data not shown).

The results in Example 9.5 show that GFP-NLS is successfully deliveredto the nucleus of HeLa cells in the presence of the shuttle agentHis-CM18-PTD4 by 2 minutes.

9.6 GFP-NLS and mCherry™-NLS Co-Transduction by His-CM18-PTD4 in HeLaCells: Visualization by Microscopy

mCherry™-NLS recombinant protein was constructed, expressed and purifiedfrom a bacterial expression system as described in Example 1.4. Thesequence of the mCherry™-NLS recombinant protein was:

[SEQ ID NO: 73] MHHHHHHGGGGSGGGGSGGASTGIRHVSKCEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGTQTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFEDGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGQVMQKKTMGWEASSERMYPEDGALKGEIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAEGRHSTGGMDELYKGGSGGGSGGGSGWIRASSGGR SSDDEATADSQHAAPPKKKRKV GG SGGGSGGGSGGGRGTEIS(MW = 34.71 kDa; pl = 6.68) NLS sequence is underlinedSerine/glycine rich linkers are in bold

GFP-NLS recombinant protein (5 μM; see Example 5.1) and mCherry™-NLSrecombinant protein (5 μM) were co-incubated together with 35 μM ofHis-CM18-PTD4, and then exposed to HeLa cells for 10 seconds usingProtocol B as described in Example 9.1. After washing steps, the cellswere immediately visualized by bright field and fluorescence microscopyat 20× magnifications as described in Example 3.2. Sample results areshown in FIG. 28, in which corresponding images showing bright field(FIG. 28A), DAPI fluorescence (FIG. 28B), GFP-NLS fluorescence (FIG.28C), and mCherry™-NLS fluorescence (FIG. 28D) are shown. White trianglewindows indicate examples of areas of co-labelling between GFP-NLS andmCherry™ fluorescence signals in cell nuclei. No significant cellularGFP or mCherry™ fluorescence was observed in negative control samples(i.e., cells exposed to GFP-NLS or mCherry™ without any shuttle agent;data not shown). These results show that GFP-NLS and mCherry™-NLS aresuccessfully delivered together to the nucleus in HeLa cells in thepresence of the shuttle agent His-CM18-PTD4.

9.7 GFP-NLS Transduction by His-CM18-PTD4 in THP-1 Suspension Cells:Flow Cytometry

The ability of the His-CM18-PTD4 to deliver GFP-NLS in the nuclei ofsuspension cells was tested using THP-1 cells. THP-1 cells were culturedand tested in the protein transduction assays using Protocols A and C asdescribed in Example 9.1, GFP-NLS (5 μM; see Example 5.1) wasco-incubated with 1 μM of His-CM18-PTD4 and exposed to THP-1 cells for 1hour (Protocol A), or was co-incubated with 5 μM of His-CM18-PTD4 andexposed to THP-1 cells for 15 seconds (Protocol C). The cells weresubjected to flow cytometry analysis as described in Example 3.3.Results are shown in Table 9.3 and in FIG. 31.

TABLE 9.3 Data from FIG. 31 Mean % Cell Conc. cells with viability Conc.of GFP signal (%) of GFP- (± St. (± St. Pro- shuttle NLS Dev.; Dev.;tocol Shuttle Cells (μM) (μM) n = 3) n = 3) C No shuttle (“Ctrl”) THP-10 5  0.2 ± 0.03 99.1 ± 0.7 A His-CM18-PTD4 1 5 14.2 ± 2.2  96.9 ± 3.6 CHis-CM18-PTD4 0.5 5 34.9 ± 3.8  82.1 ± 2.7 5 5 64.1 ± 1.6  64.0 ± 4.1

9.8 GFP-NLS Transduction by His-CM18-PTD4 in THP-1 Cells: Visualizationby Microscopy

GFP-NLS recombinant protein (5 μM; see Example 5.1) was co-incubatedwith 5 μM of His-CM18-PTD4, and then exposed to THP-1 cells for 15seconds using Protocol C as described in Example 9.1. The cells weresubjected to microscopy visualization as described in Example 3.2.

For the sample results shown in FIG. 32, GFP fluorescence of the HeLacells was immediately visualized by bright field (upper panels) andfluorescence (lower panels) microscopy at 4×, 10× and 40× magnifications(FIG. 32A-32C, respectively) after the final washing step. Whitetriangle windows in FIG. 32C indicate examples of areas of co-labellingbetween bright field and fluorescence images. FIG. 32D shows typicalresults of a corresponding FACS analysis (performed as described inExample 3.3), which indicates the percentage of cells in a 96-plate wellwith a GFP signal. Additional results are shown in FIG. 33, in whichFIGS. 33A and 33B show bright field images, and FIGS. 33C and 33D showcorresponding fluorescence images. White triangle windows indicateexamples of areas of co-labelling between FIGS. 33A and 33C, as well asFIGS. 33B and 33D. The right-most panel shows typical results of acorresponding FACS analysis (performed as described in Example 3.3),which indicates the percentage of cells in a 96-plate well with a GFPsignal.

No significant cellular GFP fluorescence was observed in negativecontrol samples (i.e., cells exposed to GFP-NLS without any shuttleagent; data not shown).

The results in this example show that GFP-NLS is successfully deliveredintracellularly in THP-1 cells in the presence of the shuttle agentHis-CM18-PTD4.

Example 10 Different Multi-Domain Shuttle Agents, but not Single-DomainPeptides, Successfully Transduce GFP-NLS in HeLa and THP-1 Cells 10.1GFP-NLS Transduction by Different Shuttle Agents in HeLa Cells: FlowCytometry

HeLa cells were cultured and tested in the protein transduction assaysusing Protocol B as described in Example 9.1. Briefly, GFP-NLSrecombinant protein (5 μM; see Example 5.1) was co-incubated with 50 μMof different shuttle agents and exposed to the HeLa cells for 10seconds. The cells were subjected to flow cytometry analysis asdescribed in Example 3.3. Results are shown in Table 10.1 and FIG. 29A.“Pos cells (%)” is the mean percentages of all cells that emanate a GFPsignal. The negative control (“Ctrl”) corresponds to cells that wereincubated with GFP-NLS recombinant protein (5 μM) without any shuttleagent.

TABLE 10.1 Data from FIG. 29A Conc. of Mean % cells with Cell viabilityConc. of GFP- GFP signal (%) shuttle NLS (± St. Dev.; (± St. Dev.;Protocol Shuttle agent Cells (μM) (μM) n = 3) n = 3) B No shuttle(“ctrl”) HeLa  0 5 0 100 His-CM18-TAT HeLa 50 55.5 ± 3.6  35.2 ± 5.7 His-CM18- HeLa 33.2 ± 2.8  41.3 ± 3.3  Transportan (TPT) TAT-KALA HeLa56.3 ± 3.6  95.6 ± 4.3  His-CM18-PTD4 HeLa   68 ± 2.2    92 ± 3.6 His-CM18-9Arg HeLa 57.2 ± 3.9  45.8 ± 5.4  TAT-CM18 HeLa 39.4 ± 3.9 23.5 ± 1.1  His-C(LLKK)₃C-PTD4 HeLa   76 ± 3.8    95 ± 2.7 His-LAH4-PTD4 HeLa   63 ± 1.64   98 ± 1.5  PTD4-KALA HeLa 73.4 ± 4.1291.4 ± 3.67 * His-LAH4-PTD4: the intracellular GFP fluorescence patternwas observed by fluorescence microscopy as being punctate, suggestingthat the GFP cargo remained trapped in endosomes.10.2 GFP-NLS Transduction by Different Shuttle Agents with VaryingIncubation Times in HeLa Cells: Flow Cytometry

HeLa cells were cultured and tested in the protein transduction assaysusing Protocol B as described in Example 9.1. Briefly, GFP-NLSrecombinant protein (5 μM; see Example 5.1) was co-incubated with 10 μMof TAT-KALA, His-CM18-PTD4, or His-C(LLKK)₃C-PTD4 for 1, 2, or 5minutes. After the final washing step, the cells were subjected to flowcytometry analysis as described in Example 3.3. Results are shown inTable 10.2 and FIG. 29B. “Pos cells (%)” is the mean percentages of allcells that emanate a GFP signal. The negative control (“Ctrl”)corresponds to cells that were incubated with GFP-NLS recombinantprotein (5 μM) without any shuttle agent.

TABLE 10.2 Data from FIG. 29B Mean % cells with Cell viability Conc. ofGFP signal (%) shuttle Incubation (± St. Dev.; (± St Dev.; ProtocolShuttle agent Cells (μM) time n = 3) n = 3) — No shuttle (“Ctrl”) HeLa 0 5 min. 0 ± n/a 97.5 ± 1.7 B TAT-KALA HeLa 10 1 min. 83.7 ± 3.5 93.5 ±2.7 2 min. 86.2 ± 4.3 92.1 ± 3.1 5 min. 68.1 ± 3.0   86 ± 4.4His-CM18-PTD4 HeLa 10 1 min. 50.6 ± 3.5 97.6 ± 2.7 2 min.   74 ± 3.380.9 ± 3.2 5 min. 82.7 ± 5.0 66.2 ± 4.4 His-C(LLKK)₃C-PTD4 HeLa 10 1min. 51.1 ± 3.5 99.5 ± 2.7 2 min. 77.8 ± 4.3 94.3 ± 3.2 5 min. 86.4 ±4.0 80.8 ± 4.410.3 GFP-NLS Transduction by TAT-KALA, His-CM18-PTD4 andHis-C(LLKK)₃C-PTD4 with Varying Incubation Times in HeLa Cells: FlowCytometry

HeLa cells were cultured and tested in the protein transduction assaysusing Protocol C as described in Example 9.1. Briefly, GFP-NLSrecombinant protein (5 μM; see Example 5.1) was co-incubated with 5 μMof TAT-KALA, His-CM18-PTD4, or His-C(LLKK)₃C-PTD4 for 1, 2, or 5minutes. After the final washing step, the cells were subjected to flowcytometry analysis as described in Example 3.3. Results are shown inTable 10.3 and FIG. 29C. The negative control (“Ctrl”) corresponds tocells that were incubated with GFP-NLS recombinant protein (5 μM)without any shuttle agent.

TABLE 10.3 Data from FIG. 29C Relative Conc. of fluorescence shuttleIncubation intensity (FL1-A) Protocol Shuttle agent Cells (μM) time (n =3) St. Dev. No shuttle (“Ctrl”) 0 5 min. 8903 501 C TAT-KALA HeLa 10 1min. 216 367 13 863.48 2 min. 506 158 14 536.28 5 min.  78 010  2 463.96His-CM18-PTD4 HeLa 10 1 min. 524 151 12 366.48 2 min. 755 624 26 933.165 min. 173 930 15 567.33 His-C(LLKK)₃C-PTD4 HeLa 10 1 min. 208 968 23669.19 2 min. 262 411.5 19 836.84 5 min. 129 890 16 693.29

10.4 GFP-NLS Transduction by Different Shuttle Agents in HeLa Cells:Flow Cytometry

HeLa cells were cultured and tested in the protein transduction assaysusing Protocol B as described in Example 9.1. Briefly, GFP-NLSrecombinant protein (5 μM; see Example 5.1) was co-incubated with 50 μMof different shuttle agents (see Table 1.3 for amino acid sequences andproperties) and exposed to the HeLa cells for 10 seconds. The cells weresubjected to flow cytometry analysis as described in Example 3.3.Results are shown in Tables 10.3a & 10.3b and FIGS. 29E & 29F. “Poscells (%)” is the mean percentages of all cells that emanate a GFPsignal. The negative control (“Ctrl”) corresponds to cells that wereincubated with GFP-NLS recombinant protein (5 μM) without any shuttleagent.

TABLE 10.3a Data from FIG. 29E Conc. Mean % Cell Conc. of cells withviability of GFP- GFP signal (%) Domain shuttle NLS (±St. Dev.; (±St.Dev., structure Shuttle agent (μM) (μM) n = 3) n = 3) — No shuttle(“Ctrl”) 0 5 0 100 ELD-CPD VSVG-PTD4 50 5 3.5 ± 1.1 100 EB1-PTD4 75.8 ±8.26  39 ± 5.6 JST-PTD4 0.84 ± 0.69 98.9 ± 0.57 His-ELD- His-C(LLKK)₃C-50 5  76 ± 3.8  95 ± 2.7 CPD PTD4 His-LAH4-PTD4*   63 ± 1.64  98 ± 1.5His-CM18-PTD4  68 ± 2.2  92 ± 3.6 His-CM18-TAT 55.5 ± 3.6  35.2 ± 5.7 His-CM18-TAT- 49.3 ± 4.1  41.4 ± 3.91 Cys** His-CM18-9Arg 57.2 ± 3.9345.8 ± 3.53 His-CM18- 33.2 ± 2.82 41.3 ± 3.29 Transportan (TPT)*His-LAH4-PTD4: the intracellular GFP fluorescence pattern was observedby fluorescence microscopy as being punctate, suggesting that the GFPcargo remained trapped in endosomes. **Not shown in FIG. 29E.

TABLE 10.3b Data from FIG. 29F Conc. Mean % Cell Conc. of cells withviability of GFP- GFP signal (%) Domain shuttle NLS (±St. Dev.; (±St.Dev.; structure Shuttle agent (μM) (μM) n = 3) n = 3) — No shuttle(“Ctrl”) 0 5 0 100 CPD-ELD TAT-CM18 50 5 39.4 ± 3.9  23.5 ± 1.1 TAT-KALA 56.3 ± 3.6  95.6 ± 4.3  PTD4-KALA 73.4 ± 4.12 91.4 ± 3.679Arg-KALA  7.8 ± 1.53 62.8 ± 5.11 Pep1-KALA 17.2 ± 3.07 94.7 ± 3.77Xentry-KALA 19.4 ± 1.01 98.3 ± 0.64 SynB3-KALA 14.3 ± 2.37 91.1 ± 0.82

HeLa cells were cultured and tested in the protein transduction assaysusing Protocol B as described in Example 9.1. Briefly, GFP-NLSrecombinant protein (5 μM; see Example 5.1) was co-incubated with 10 μMof TAT-KALA, His-CM18-PTD4, or His-C(LLKK)₃C-PTD4 for 1, 2, or 5minutes. After the final washing step, the cells were subjected to flowcytometry analysis as described in Example 3.3. Results are shown inTables 10.3c & 10.3b and FIGS. 29G and 29H. “Pos cells (%)” is the meanpercentages of all cells that emanate a GFP signal. The negative control(“Ctrl”) corresponds to cells that were incubated with GFP-NLSrecombinant protein (5 μM) without any shuttle agent.

TABLE 10.3c Data from FIG. 29G Mean % cells Cell Conc. of with GFPviability Conc. of GFP- Incubation signal (%) Domain shuttle NLS time (±St. Dev.; (± St. Dev.; structure Shuttle agent (μM) (μM) (min) n = 3) n= 3) — No shuttle (“Ctrl”)  0 5 5   0 ± n/a 98.3 ± 0.9 1 64.6 ± 4.3 96.2± 3.0 CPD-ELD PTD4-KALA 10 5 2 78.8 ± 3.6 75.3 ± 3.8 5 71.4 ± 4.2 82.4 ±4.7 ELD-CPD EB1-PTD4 10 5 1 76.3 ± 3.5 61.7 ± 2.7 2 79.0 ± 3.3 56.6 ±3.2 5 71.1 ± 5.0 55.8 ± 4.4 His-ELD-CPD- His-CM18- 10 5 1 68.6 ± 3.568.1 ± 2.7 His PTD4-His 2 74.1 ± 4.3 61.6 ± 3.2 5 59.8 ± 4.0 41.2 ± 4.4

TABLE 10.3d Data from FIG. 29H Relative Conc. Fluorescence Conc. ofIntensity of GFP- Incubation (FL1-A) Domain Shuttle shuttle NLS time(±St. Dev.; structure agent (μM) (μM) (min) n = 3) — No shuttle 0 5 5  8903 ± 501.37 (“Ctrl”) CPD-ELD PTD4-KALA 10 5 1 190 287 ± 9445   2 386480 ± 17 229 5 241 230 ± 14 229 ELD-CPD EB1-PTD4 10 5 1 178 000 ± 11 9342 277 476 ± 25 319 5 376 555 ± 16 075 His-ELD- His-CM18- 10 5 1 204 338± 22 673 CPD-His PTD4-His 2 307 329 ± 19 618 5 619 964 ± 17 411

The shuttle agent CM18-PTD4 was used as a model to demonstrate themodular nature of the individual protein domains, as well as theirability to be modified. More particularly, the presence or absence of:an N-terminal cysteine residue (“Cys”); different flexible linkersbetween the ELD and CPD domains (“L1”: GGS; “L2”: GGSGGGS; and “L3”:GGSGGGSGGGS) and different lengths, positions, and variants tohistidine-rich domains; were studied.

HeLa cells were cultured and tested in the protein transduction assaysusing Protocol B as described in Example 9.1. Briefly, GFP-NLSrecombinant protein (5 μM; see Example 5.1) was co-incubated with 20 μMof different shuttle peptide variants (see Table 1.3 for amino acidsequences and properties) of the shuttle agent His-CM18-PTD4 for 1minute. After the final washing step, the cells were subjected to flowcytometry analysis as described in Example 3.3. Results are shown inTable 10.3e and FIG. 29I. “Pos cells (%)” is the mean percentages of allcells that emanate a GFP signal. The negative control (“Ctrl”)corresponds to cells that were incubated with GFP-NLS recombinantprotein (5 μM) without any shuttle agent

TABLE 10.3e Data from FIG. 29I Conc. Mean % Cell Conc. of cells withviability of GFP- GFP signal (%) Domain shuttle NLS (±St Dev.; (±St.Dev.; structure Shuttle agent (μM) (μM) n = 3) n = 3) — No shuttle 0 5 0 99.6 ± 0.12 (“Ctrl”) ELD-CPD CM18-PTD4 47.6 ± 2.6 33.9 ± 3.7 Cys-CM18-36.6 ± 2.3 78.7 ± 3.1 PTD4 CM18-L1-PTD4 20 5 48.5 ± 3.0 50.1 ± 3.8CM18-L2-PTD4 45.5 ± 6.5 64.0 ± 1.3 CM18-L3-PTD4 39.0 ± 2.7 71.9 ± 6.0His-ELD- His-CM18-PTD4 20 5 60.3 ± 3.2 81.6 ± 4.5 CPD His-CM18-  41.3 ±4.28   62 ± 5.76 PTD4-6Cys Met-His-CM18-  45.6 ± 3.88  54.9 ± 3.45PTD4-Cys 3His-CM18-PTD4 39.4 ± 0.5 39.2 ± 3.3 12His-CM18- 36.9 ± 4.333.4 ± 4.3 PTD4 HA-CM18-PTD4 42.3 ± 4.2 68.3 ± 4.1 3HA-CM18-PTD4 37.2 ±3.9 43.6 ± 2.8 ELD-His- CM18-His-PTD4 20 5 61.7 ± 1.8 57.7 ± 4.2 CPDHis-ELD- His-CM18-PTD4- 20 5 68.0 ± 6.0 78.6 ± 1.1 CPD-His His

These results show that variations in a given shuttle (e.g., CM18-PTD4)may be used to modulate the degree of transduction efficiency and cellviability of the given shuttle. More particularly, the addition of anN-terminal cysteine residue to CM18-PTD4 (see Cys-CM18-PTD4), decreasedGFP-NLS transduction efficiency by 11% (from 47.6% to 36.6%), butincreased cell viability from 33.9% to 78.7%. Introduction of flexiblelinker domains (L1, L2, and L3) of different lengths between the CM18and PTD4 domains did not result in a dramatic loss of transductionefficiency, but increased cell viability (see CM18-L1-PTD4,CM18-L2-PTD4, and CM18-L3-PTD4). Finally, variations to the amino acidsequences and/or positions of the histidine-rich domain(s) did notresult in a complete loss of transduction efficiency and cell viabilityof His-CM18-PTD4 (see 3His-CM18-PTD4, 12His-CM18-PTD4, HA-CM18-PTD4,3HA-CM18-PTD4, CM18-His-PTD4, and His-CM18-PTD4-His). Of note, adding asecond histidine-rich domain at the C terminus of His-CM18-PTD4 (i.e.,His-CM18-PTD4-His) increased transduction efficiency from 60% to 68%with similar cell viability.

10.5 Lack of GFP-NLS Transduction by Single-Domain Peptides or a His-CPDPeptide in HeLa Cells: Flow Cytometry

HeLa cells were cultured and tested in the protein transduction assaysusing Protocol B as described in Example 9.1. Briefly, GFP-NLSrecombinant protein (5 μM; see Example 5.1) was co-incubated with 50 μMof different single-domain peptides (TAT; PTD4; Penetratin; CM18;C(LLKK)₃C; KALA) or the two-domain peptide His-PTD4 (lacking an ELD),and exposed to the HeLa cells for 10 seconds. After the final washingstep, the cells were subjected to flow cytometry analysis as describedin Example 3.3. Results are shown in Table 10.4 and FIG. 29D. “Pos cells(%)” is the mean percentages of all cells that emanate a GFP signal. Thenegative control (“Ctrl”) corresponds to cells that were incubated withGFP-NLS recombinant protein (5 μM) without any single-domain peptide orshuttle agent

TABLE 10.4 Data from FIG. 29D Mean % cells with GFP Cell viability Conc.of Conc. of signal (%) Single-domain shuttle GFP-NLS (± St. Dev.; (± St.Dev.; Protocol Domain peptide Cells (μM) (μM) n = 3) n = 3) B — Nopeptide HeLa  0 5  0.1 ± 0.02 98.3 ± 0.59 (“Ctrl”) CPD TAT HeLa 50 5 1.1 ± 0.27 94.6 ± 0.44 PTD4  1.1 ± 0.06   94 ± 4.5  Penetratin  3.6 ±0.1    96 ± 0.6  (Pen) ELD CM18 HeLa 50 5  2.9 ± 0.2    95 ± 1.2 C(LLKK)₃C  1.1 ± 0.57 61.8 ± 0.1  KALA  1.4 ± 0.13   84 ± 0.7  His-CPDHis-PTD4 HeLa 50 5 1.04 ± 0.12 96.5 ± 0.28

These results show that the single-domain peptides TAT, PTD4,Penetratin, CM18, C(LLKK)₃C, KALA, or the two-domain peptide His-PTD4(lacking an ELD), are not able to successfully transduce GFP-NLS in HeLacells.

10.6 GFP-NLS Transduction by TAT-KALA, His-CM18-PTD4, His-C(LLKK),C-PTD4, PTD4-KALA, EB1-PTD4, and His-CM18-PTD4-Hs in HeLa Cells:Visualization by Microscopy

GFP-NLS recombinant protein (5 μM; see Example 5.1) was co-incubatedwith 50 μM of shuttle agent, and then exposed to HeLa cells for 10seconds using Protocol B as described in Example 9.1. The cells werevisualized by microscopy as described in Example 3.2, after anincubation time of 2 minutes.

For the sample results shown in FIG. 30, GFP fluorescence of the HeLacells was immediately visualized by bright field (bottom row panels) andfluorescence (upper and middle row panels) microscopy at 20× or 40×magnifications after the final washing step. The results with theshuttle agents TAT-KALA, His-CM18-PTD4, and His-C(LLKK)₃C-PTD4 are shownin FIGS. 30A, 30B and 30C, respectively. The results with the shuttleagents PTD4-KALA, EB1-PTD4, and His-CM18-PTD4-His are shown in FIGS.30D, 30E and 30F, respectively. The insets in the bottom row panels showthe results of corresponding FACS analyses (performed as described inExample 3.3), which indicates the percentage of cells in a 96-plate wellwith a GFP signal. No significant cellular GFP fluorescence was observedin negative control samples (i.e., cells exposed to GFP-NLS without anyshuttle agent; data not shown).

10.7 GFP-NLS Transduction by TAT-KALA, His-CM18-PTD4 and His-C(LLKK)C-PTD4 with Varying Incubation Times in THP-1 Cells: Flow Cytometry

THP-1 cells were cultured and tested in the protein transduction assaysusing Protocol C as described in Example 9.1. Briefly, GFP-NLSrecombinant protein (5 μM; see Example 5.1) was co-incubated with 1 μMof TAT-KALA, His-CM18-PTD4, or His-C(LLKK)₃C-PTD4 for 15, 30, 60, or 120seconds. After the final washing step, the cells were subjected to flowcytometry analysis as described in Example 3.3. The mean percentages ofcells emanating a GFP signal (“Pos cells (%)”) are shown in Table 10.4aand in FIG. 34A. The mean fluorescence intensity is shown in Table 10.5and FIG. 34B. The negative control (“Ctrl”) corresponds to cells thatwere incubated with GFP-NLS recombinant protein (5 μM) without anyshuttle agent

TABLE 10.4a Data from FIG. 34A Conc. Mean % Cell Conc. of cells withviability of GFP- GFP signal (%) shuttle NLS Incubation (±St. Dev.;(±St. Dev.; Protocol Shuttle agent Cells (μM) (μM) time (sec.) n = 3) n= 3) C No shuttle (“Ctrl”) THP-1 0 5 120  1.12 ± 0.27  97.3 ± 1.55TAT-KALA THP-1 1 5 15   47 ± 3.5 84.6 ± 2.7 30 52.9 ± 1.3 70.3 ± 3.2 6070.1 ± 2.0 82.7 ± 1.4 120 82.1 ± 2.5 46.3 ± 4.9 His-CM18-PTD4 THP-1 1 515 23.7 ± 0.2   90 ± 3.0 30   53 ± 0.3   89 ± 1.1 60 69.6 ± 4.2 85.3 ±3.6 120   89 ± 0.8 74.3 ± 3.2 His-C(LLKK)₃C- THP-1 1 5 15 38.4 ± 0.385.2 ± 2.8 PTD4 30 42.3 ± 4.2   86 ± 2.0 60 64.5 ± 1.0 86.9 ± 3.8 12078.7 ± 0.3 79.6 ± 2.8

TABLE 10.5 Data from FIG. 34B Relative Conc. Incu- fluorescence ofbation intensity shuttle time (FL1-A) Standard Protocol Shuttle agentCells (μM) (sec.) (n = 3) Deviation C No shuttle THP- 0 120 217   23.09(“Ctrl”) 1 TAT-KALA THP- 1 15 6 455.12 333.48 1 30 8 106.81 436.28 60 13286.2  463.96 120 27 464.92  2 366.48   His-CM18- THP- 1 15 5 605.45933.16 PTD4 1 30 25 076.41  5 567.33   60 34 046.94  3 669.19   120 55613.48  9 836.84   His- THP- 1 15 5 475.12 693.29 C(LLKK)₃C- 1 30 5755.8  635.18 PTD4 60 8 267.38 733.29 120 21 165.06  209.37

Example 11 Repeated Daily Treatments with Low Concentrations of ShuttleAgent in the Presence of Serum Results in GFP-NLS Transduction in THP-1Cells

11.1 GFP-NLS Transduction with His-CM18-PTD4 or His-C(LLKK)3C-PTD4 inTHP-1 Cells: Flow Cytometry

THP-1 cells were cultured and tested in the protein transduction assayusing Protocol A as described in Example 9.1, but with the followingmodifications. GFP-NLS recombinant protein (5, 2.5, or 1 μM; see Example5.1) was co-incubated with 0.5 or 0.8 μM of His-CM18-PTD4, or with 0.8μM of His-C(LLKK)₃C-PTD4, and then exposed to THP-1 cells each day for150 min in the presence of cell culture medium containing serum. Cellswere washed and subjected to flow cytometry analysis as described inExample 3.3 after 1 or 3 days of repeated exposure to the shuttleagent/cargo. The results are shown in Table 11.1 and in FIGS. 35A, 358,35C and 35F. The negative control (“Ctrl”) corresponds to cells thatwere incubated with GFP-NLS recombinant protein (5 μM) without anyshuttle agent.

TABLE 11.1 Data from FIG. 35A, 35B, 35C and 35F Conc. of Conc. ofExposure to Mean % cells shuttle GFP-NLS shuttle/cargo with GFP signalCell viability (%) FIG. Shuttle agent Cells (μM) (μM) (days) (±St. Dev.;n = 3) (±St. Dev.; n = 3) 35A No shuttle (Ctrl) THP-1 0 5 0 0.15 ± 0.0498.7 ± 0.1 His-CM18-PTD4 0.5 5 1 12.1 ± 1.5  98.2 ± 2.4 3 73.4 ± 1.1 84.3 ± 3.8 35B No shuttle (Ctrl) THP-1 0 5 0 0.36 ± 0.09 97.1 ± 1.2His-CM18-PTD4 0.8 2.5 1 12.2 ± 0.9  92.3 ± 1.9 3 62.4 ± 3.5  68.5 ± 2.235C No shuttle (Ctrl) THP-1 0 5 0 0.28 ± 0.05 96.4 ± 2.0 His-CM18-PTD40.8 1 1 1.6 ± 0.2 98.4 ± 6.4 3 6.5 ± 0.9 80.6 ± 4.6 35F No shuttle(Ctrl) THP-1 0 5 0 0.62 ± 0.11 96.3 ± 1.4 His-C(LLKK)₃C- 0.8 1 1 1.8 ±0.2 97.2 ± 2.2 PTD4 3 6.6 ± 0.8 76.6 ± 3.4

The viability of THP-1 cells repeatedly exposed to His-CM18-PTD4 andGFP-NLS was determined as described in Example 3.3a. The results areshown in Tables 11.2 and 11.3 and in FIGS. 35D and 35E. The results inTable 11.2 and FIG. 35D show the metabolic activity index of the THP-1cells after 1, 2, 4, and 24 h, and the results in Table 11.3 and FIG.35E show the metabolic activity index of the THP-1 cells after 1 to 4days.

TABLE 11.2 Data from FIG. 35D Mean metabolic activity index Conc. ofConc. of (±St. Dev.; n = 3) shuttle GFP-NLS (Exposure to shuttle/cargo)Shuftle agent Cells (μM) (μM) 1 h 2 h 4 h 24 h No shuttle (Ctrl) THP-1 05 40810 ± 38223 ± 44058 ± 42362 ± 757.39 238.66 320.23 333.80His-CM18-PTD4 THP-1 0.5 5  9974 ±  9707 ±  3619 ±  2559 ± 1749.85 1259.82  2247.54  528.50 1 5 42915 ± 41386 ± 44806 ± 43112 ± 259.67670.66 824.71 634.56

TABLE 11.3 Data from FIG. 35E Mean metabolic activity index Conc. ofConc. of (±St. Dev.; n = 3) shuttle GFF-NLS (Exposure to shuttle/cargo)Shuttle agent Cells (μM) (μM) 1 day 2 days 3 days 4 days No shuttle(Ctrl) THP-1 0 5 44684 ± 43389 ± 45312 ± 43697 ± 283.27 642,47 963.401233    0.5 5 44665 ± 42664 ± 43927 ± 43919 ± 310.3  398.46 3511.54 4452.25  His-CM184FTD4 THP-1 0.8 5 44531 ± 43667 ± 44586 ± 44122 ±176.66 421.66 383.68 239.98 1 5 41386 ± 36422 ± 27965 ± 22564 ± 670.66495.01 165.33 931.28

The results in Example 11 show that repeated daily (or chronic)treatments with relatively low concentrations of His-CM18-PTD4 orHis-C(LLKK)₃C-PTD4 in the presence of serum result in intracellulardelivery of GFP-NLS in THP-1 cells. The results also suggest that thedosages of the shuttle agents and the cargo can be independentlyadjusted to improve cargo transduction efficiency and/or cell viability.

Example 12 His-CM18-PTD4 Increases Transduction Efficiency and NuclearDelivery of GFP-NLS in a Plurality of Cell Lines

12.1 GFP-NLS Transduction with His-CM18-PTD4 in Different Adherent &Suspension Cells: Flow Cytometry

The ability of the shuttle agent His-CM18-PTD4 to deliver GFP-NLS to thenuclei of different adherent and suspension cells using Protocols B(adherent cells) or C (suspension cells) as described in Example 9.1 wasexamined. The cell lines tested included: HeLa, Balb3T3, HEK 293T, CHO,NIH3T3, Myoblasts, Jurkat, THP-1, CA46, and HT2 cells, which werecultured as described in Example 1. GFP-NLS (5 μM; see Example 5.1) wasco-incubated with 35 μM of His-CM18-PTD4 and exposed to adherent cellsfor 10 seconds (Protocol B), or was co-incubated with 5 μM ofHis-CM18-PTD4 and exposed to suspension cells for 15 seconds (ProtocolC). Cells were washed and subjected to flow cytometry analysis asdescribed in Example 3.3. Results are shown in Table 12.1 and FIG. 36.“Pos cells (%)” is the mean percentages of all cells that emanate a GFPsignal.

TABLE 12.1 Data from FIG. 36 Conc. of Conc. of Mean % cells shuttleGFP-NLS with GFP signal Cell viability (%) Shuttle agent Protocol (μM)(μM) Cells (±St. Dev.; n = 3) (±St. Dev.; n = 3) His-CM18-PTD4 B 35 5HeLa 72.3 ± 5.3 94.6 ± 0.4 Balb3T3 40.2 ± 3.1 98.4 ± 0.6 HEK 293T 55.3 ±0.2 95.3 ± 1.2 CHO 53.7 ± 4.6 92.8 ± 0.1 NIH3T3 35.4 ± 3.9  3.3 ± 5.4Myoblasts 25.6 ± 2.6 23.5 ± 1.1 C 5 5 Jurkat 30.7 ± 2.2 73.6 ± 0.7 THP-164.1 ± 1.6 64.1 ± 4.5 CA46 24.4 ± 0.6 71.6 ± 1.0 HT2 30.5 ± 2.5 90.6 ±1.5

TABLE 12.1 Data from FIG. 36 Conc. of Conc. of Mean % cells shuttleGFP-NLS with GFP signal Cell viability (%) Shuttle agent Protocol (μM)(μM) Cells (±St. Dev.; n = 3) (±St. Dev.; n = 3) His-CM18-PTD4 B 35 5HeLa 72.3 ± 5.3 94.6 ± 0.4 Balb3T3 40.2 ± 3.1 98.4 ± 0.6 HEK 293T 55.3 ±0.2 95.3 ± 1.2 CHO 53.7 ± 4.6 92.8 ± 0.1 NIH3T3 35.4 ± 3.9  3.3 ± 5.4Myoblasts 25.6 ± 2.6 23.5 ± 1.1 C 5 5 Jurkat 30.7 ± 2.2 73.6 ± 0.7 THP-164.1 ± 1.6 64.1 ± 4.5 CA46 24.4 ± 0.6 71.6 ± 1.0 HT2 30.5 ± 2.5 90.6 ±1.512.2 GFP-NLS Transduction with His-CM18-PTD4 in Several Adherent andSuspension Cells: Visualization by Microscopy

GFP-NLS recombinant protein (5 μM; see Example 5.1) was co-incubatedwith 35 μM of His-CM18-PTD4 and exposed to adherent cells for 10 secondsusing Protocol A, or was co-incubated with 5 μM of His-CM18-PTD4 andexposed to suspension cells for 15 seconds using Protocol B, asdescribed in Example 9.1. After washing the cells, GFP fluorescence wasvisualized by bright field and fluorescence microscopy. Sample imagescaptured at 10× magnifications showing GFP fluorescence are shown for293T (FIG. 37A), Balb3T3 (FIG. 37B), CHO (FIG. 37C), Myoblasts (FIG.37D), Jurkat (FIG. 37E), CA46 (FIG. 37F), HT2 (FIG. 37G), and NIH3T3(FIG. 37H) cells. The insets show corresponding flow cytometry resultsperformed as described in Example 3.3, indicating the percentage ofGFP-NLS-positive cells. No significant cellular GFP fluorescence wasobserved in negative control samples (i.e., cells exposed to GFP-NLSwithout any shuttle agent; data not shown).

Nuclear localization of the GFP-NLS was further confirmed in fixed andpermeabilized myoblasts using cell immuno-labelling as described inExample 3.2a. GFP-NLS was labeled using a primary mouse monoclonalanti-GFP antibody (Feldan, # A017) and a secondary goat anti-mouseAlexa™-594 antibody (Abcam #150116). Nuclei were labelled with DAPI.Sample results for primary human myoblast cells are shown in FIG. 38, inwhich GFP immuno-labelling is shown in FIG. 38A, and an overlay of theGFP immuno-labelling and DAPI labelling is shown in FIG. 388. Nosignificant cellular GFP labelling was observed in negative controlsamples (i.e., cells exposed to GFP-NLS without any shuttle agent; datanot shown).

The microscopy results revealed that GFP-NLS is successfully deliveredto the nucleus of all the tested cells using the shuttle agentHis-CM18-PTD4.

Example 13 His-CM18-PTD4 Enables Transduction of a CRISPR/Cas9-NLSSystem and Genome Editing in Hela Cells 13.1 Cas9-NLS RecombinantProtein

Cas9-NLS recombinant protein was constructed, expressed and purifiedfrom a bacterial expression system as described in Example 1.4. Thesequence of the Cas9-NLS recombinant protein produced was:

[SEQ ID NO: 74] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGDGGRSSDDEA TADSQHAAPPKKKRKVGGSGGGSGGGSGGGRHHHHHH (MW = 162.9 kDa; pl = 9.05)NLS sequence is underlined Serine/glycine rich linkers are in bold

13.2 Transfection Plasmid Surrogate Assay

This assay enables one to visually identify cells that have beensuccessfully delivered an active CRISPR/Cas9 complex. As shown in FIG.39A, the assay involves transfecting cells with an expression plasmidDNA encoding the fluorescent proteins mCherry™ and GFP, with a STOPcodon separating their two open reading frames. Transfection of thecells with the expression plasmid results in mCherry™ expression, but noGFP expression (FIG. 39B). A CRISPR/Cas9 complex, which has beendesigned/programmed to cleave the plasmid DNA at the STOP codon, is thendelivered intracellularly to the transfected cells expressing mCherry™(FIG. 39D). Successful transduction of an active CRISPR/Cas9 complexresults in the CRISPR/Cas9 complex cleaving the plasmid DNA at the STOPcodon (FIG. 39C). In a fraction of the cells, random non-homologous DNArepair of the cleaved plasmid occurs and results in removal of the STOPcodon, and thus GFP expression and fluorescence (FIG. 39E).

On Day 1 of the transfection plasmid surrogate assay, DNA plasmids fordifferent experimental conditions (250 ng) are diluted in DMEM (50 μL)in separate sterile 1.5-mL tubes, vortexed and briefly centrifuged. Inseparate sterile 1.5-mL tubes, Fastfect™ transfection reagent wasdiluted in DMEM (50 μL) with no serum and no antibiotics at a ratio of3:1 (3 μL of Fastfect™ transfection reagent for 1 μg of DNA) and thenquickly vortexed and briefly centrifuged. The Fastfect™/DMEM mixture wasthen added to the DNA mix and quickly vortexed and briefly centrifuged.The Fastfect™/DMEM/DNA mixture is then incubated for 15-20 min at roomtemperature, before being added to the cells (100 μL per well). Thecells are then incubated at 37° C. and 5% CO₂ for 5 h. The media is thenchanged for complete medium (with serum) and further incubated at 37° C.and 5% CO₂ for 24-48 h. The cells are then visualized under fluorescentmicroscopy to view the mCherry™ signal.

13.3 His-CM18-PTD4-Mediated CRISPR/Cas9-NLS System Delivery and Cleavageof Plasmid DNA

RNAs (crRNA & tracrRNA) were designed to target a nucleotide sequence ofthe EMX1 gene, containing a STOP codon between the mCherry™ and GFPcoding sequences in the plasmid of Example 13.2. The sequences of thecrRNA and tracrRNA used were as follows:

crRNA [SEQ ID NO: 75]: 5′-GAGUCCGAGCAGAAGAAGAAGUUUUAGAGCUAUGCUGUUUUG-3′tracrRNA [SEQ ID NO: 76]:5′-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3′

HeLa cells were cultured and subjected to the transfection plasmidsurrogate assay as described in Example 13.2). On Day 1, the HeLa cellswere transfected with a plasmid surrogate encoding the mCherry™ proteinas shown in FIG. 39A. On Day 2, a mix of Cas9-NLS recombinant protein (2μM; see Example 13.1) and RNAs (crRNA & tracrRNA; 2 μM; see above) wereco-incubated with 50 μM of His-CM18-PTD4, and the mixture (CRISPR/Cas9complex) was exposed to HeLa cells for 10 seconds using Protocol B asdescribed in Example 9.1. Double-stranded plasmid DNA cleavage by theCRISPR/Cas9 complex at the STOP codon between the mCherry™ and GFPcoding sequences (FIG. 39B), and subsequent non-homologous repair by thecell in some cases results in removal of the STOP codon (FIG. 39C),thereby allowing expression of both the mCherry™ and GFP fluorescentproteins in the same cell on Day 3 (FIG. 39D-39E). White trianglewindows in FIGS. 39D and 39E indicate examples of areas of co-labellingbetween mCherry™ and GFP.

As a positive control for the CRISPR/Cas9-NLS system, HeLa cells werecultured and co-transfected with three plasmids: the plasmid surrogate(as described in Example 13.2) and other expression plasmids encodingthe Cas9-NLS protein (Example 13.1) and the crRNA/tracrRNAs (Example13.3). Typical fluorescence microscopy results are shown in FIG. 40A-D.Panels A and B show cells 24 hours post-transfection, while panels C andD show cells 72 hours post-transfection.

FIG. 40E-40H shows the results of a parallel transfection plasmidsurrogate assay performed using 35 μM of the shuttle His-CM18-PTD4, asdescribed for FIG. 39. FIGS. 40E and 40F show cells 24 hourspost-transduction, while panels G and H show cells 48 hourspost-transduction. FIGS. 40E and 40G show mCherry™ fluorescence, andFIGS. 40F and 40H show GFP fluorescence, the latter resulting fromremoval of the STOP codon by the transduced CRISPR/Cas9-NLS complex andsubsequent non-homologous repair by the cell. No significant cellularGFP fluorescence was observed in negative control samples (i.e., cellsexposed to CRISPR/Cas9-NLS complex without any shuttle agent; data notshown).

13.4 T7E1 Assay

The T7 endonuclease I (T7E1) can be used to detect on-target CRISPR/Casgenome editing events in cultured cells. As an overview, genomic DNAfrom target cells is amplified by PCR. The PCR products are thendenatured and reannealed to allow heteroduplex formation betweenwild-type DNA and CRISPR/Cas-mutated DNA T7E1, which recognizes andcleaves mismatched DNA, is used to digest the heteroduplexes. Theresulting cleaved and full-length PCR products are visualized by gelelectrophoresis.

The T7E1 assay was performed with the Edit-R™ Synthetic crRNA PositiveControls (Dharmacon # U-007000-05) and the T7 Endonuclease I (NEB, Cat #M0302S). After the delivery of the CRISPR/Cas complex, cells were lysedin 100 μL of Phusion™ High-Fidelity DNA polymerase (NEB # M0530S)laboratory with additives. The cells were incubated for 15-30 minutes at56° C., followed by deactivation for 5 minutes at 96° C. The plate wasbriefly centrifuged to collect the liquid at bottom of the wells. 50-μLPCR samples were set up for each sample to be analyzed. The PCR sampleswere heated to 95° C. for 10 minutes and then slowly (>15 minutes)cooled to room temperature. PCR product (˜5 μL) was then separated on anagarose gel (2%) to confirm amplification. 15 μL of each reaction wasincubated with T7E1 nuclease for 25 minutes at 37° C. Immediately, theentire reaction volume was run with the appropriate gel loading bufferon an agarose gel (2%).

13.5 His-CM18-PTD4 and His-C(LLKK)₃C-PTD4-Mediated CRISPR/Cas9-NLSSystem Delivery and Cleavage of Genomic PPIB Sequence

A mix composed of a Cas9-NLS recombinant protein (25 nM; Example 13.1)and crRNA/tracrRNA (50 nM; see below) targeting a nucleotide sequence ofthe PPIB gene were co-incubated with 10 μM of His-CM18-PTD4 orHis-C(LLKK)₃C-PTD4, and incubated with HeLa cells for 16 h in mediumwithout serum using Protocol A as described in Example 9.1.

The sequences of the crRNA and tracrRNAs constructed and their targetswere:

Feldan tracrRNA [SEQ ID NO: 77]:5′-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3′ PPIB crRNA [SEQ ID NO: 78]:5′-GUGUAUUUUGACCUACGAAUGUUUUAGAGCUAUGCUGUUUUG-3′Dharmacon tracrRNA [SEQ ID NO: 79]:5′-AACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU-3′

After 16 h, HeLa cells were washed with PBS and incubated in medium withserum for 48 h. HeLa cells were harvested to proceed with the T7E1protocol assay as described in Example 13.4.

FIG. 41A shows an agarose gel with the PPIB DNA sequences after PCRamplification. Lane A shows the amplified PPIB DNA sequence in HeLacells without any treatment (i.e., no shuttle or Cas9/RNAs complex).Lanes B: The two bands framed in white box #1 are the cleavage productof the PPIB DNA sequence by the CRIPR/Cas9 complex after the delivery ofthe complex with the shuttle His-C(LLKK)₃C-PTD4. Lane C: These bandsshow the amplified PPIB DNA sequence after incubation of the HeLa cellswith the Cas9/RNAs complex without shuttle (negative control). Lane D:The bands framed in white box #2 show the amplified PPIB DNA sequenceafter incubation of the HeLa cells with the Cas9/RNAs complex inpresence of a lipidic transfection agent (DharmaFect™ transfectionreagent # T-20XX-01) (positive control). Similar results were obtainedusing the shuttle His-CM18-PTD4 (data not shown).

FIG. 41B shows an agarose gel with the PPIB DNA sequences after PCRamplification. The left panel shows the cleavage product of theamplified PPIB DNA sequence by the CRIPR/Cas9 complex after the deliveryof the complex with the shuttle agent His-CM18-PTD4 in HeLa cells. Theright panel shows amplified DNA sequence before the T7E1 digestionprocedure as a negative control.

FIG. 41C shows an agarose gel with the PPIB DNA sequences after PCRamplification. The left panel shows the amplified PPIB DNA sequenceafter incubation of the HeLa cells with the Cas9/RNAs complex inpresence of a lipidic transfection agent (DharmaFect™ transfectionreagent # T-20XX-1) (positive control). The right panel shows amplifiedDNA sequence before the T7E1 digestion procedure as a negative control.

These results show that the shuttle agents His-CM18-PTD4 andHis-C(LLKK)₃C-PTD4 successfully deliver a functional CRISPR/Cas9 complexto the nucleus of HeLa cells, and that this delivery results inCRISPR/Cas9-mediated cleavage of genomic DNA.

13.6 CRISPR/Cas9-NLS System Delivery by Different Shuttle Agents, andCleavage of Genomic HPTR Sequence in HeLa and Jurkat Cells

A mix composed of a Cas9-NLS recombinant protein (2.5 μM; Example 13.1)and crRNA/tracrRNA (2 μM; see below) targeting a nucleotide sequence ofthe HPTR gene were co-incubated with 35 μM of His-CM18-PTD4,His-CM18-PTD4-His, His-C(LLKK)3C-PTD4, or EB1-PTD4, and incubated withHeLa or Jurkat cells for 2 minutes in PBS using Protocol B as describedin Example 9.1.

The sequences of the crRNA and tracrRNAs constructed and their targetswere:

Feldan tracrRNA [SEQ ID NO: 77]: 5′-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3′ HPRT crRNA [SEQ ID NO: 103]:5′-AAUUAUGGGGAUUACUAGGAGUUUUAGAGCUAUGCU-3′

After 2 minutes, cells were washed with PBS and incubated in medium withserum for 48 h. Cells were harvested to proceed with the T7E1 protocolassay as described in Example 13.4. FIG. 46 shows an agarose gel withthe HPTR DNA sequences after PCR amplification and the cleavage productof the amplified HPTR DNA sequence by the CRISPR/Cas9 complex after thedelivery of the complex with the different shuttle agents. FIG. 46Ashows the results with the shuttle agents: His-CM18-PTD4,His-CM18-PTD4-His, and His-C(LLKK)3C-PTD4 in HeLa cells. FIG. 46B showsthe results with His-CM18-PTD4 and His-CM18-L2-PTD4 in Jurkat cells.Negative controls (lanes 4) show amplified HPTR DNA sequence afterincubation of the cells with the CRISPR/Cas9 complex without thepresence of the shuttle agent. Positive controls (lane 5 in FIGS. 46Aand 46B) show the amplified HPTR DNA sequence after incubation of thecells with the Cas9/RNAs complex in presence of a lipidic transfectionagent (Lipofectamine® RNAiMAX™ Transfection Reagent ThermoFisher ProductNo. 13778100).

These results show that different polypeptide shuttle agents of thepresent description may successfully deliver a functional CRISPR/Cas9complex to the nucleus of HeLa and Jurkat cells, and that this deliveryresults in CRISPR/Cas9-mediated cleavage of genomic DNA.

Example 14 His-CM18-PTD4 Enables Transduction of the TranscriptionFactor HOXB4 in THP-1 Cells 14.1 HOXB4-WT Recombinant Protein

Human HOXB4 recombinant protein was constructed, expressed and purifiedfrom a bacterial expression system as described in Example 1.4. Thesequence of the HOXB4-WT recombinant protein produced was:

[SEQ ID NO: 80] MHHHHHHMAMSSFLINSNYVDPKFPPCEEYSQSDYLPSDHSPGYYAGGQRRESSFQPEAGFGRRAACTVQRYPPPPPPPPPPGLSPRAPAPPPAGALLPEPGQRCEAVSSSPPPPPCAQNPLHPSPSHSACKEPVVYPWMRKVHVSTVNPNYAGGEPKRSRTAYTRQQVLELEKEFHYNRYLTRRRRVEIAHALCLSERQIKIWFQNRRMKWKKDHKLPNTKIRSGGAAGSAGGPPGRPNGGPRAL(MW = 28.54 kDa; pI = 9.89)The initiator methionine and the 6x Histidine tag are shown in bold.14.2 Real-Time Polymerase Chain Reaction (rt-PCR)

Control and treated cells are transferred to separate sterile 1.5-mLtubes and centrifuged for 5 minutes at 300 g. The cell pellets areresuspended in appropriate buffer to lyse the cells. RNAase-free 70%ethanol is then added followed by mixing by pipetting. The lysates aretransferred to an RNeasy™ Mini spin column and centrifuged 30 seconds at13000 RPM. After several washes with appropriate buffers andcentrifugation steps, the eluates are collected in sterile 1.5-mL tubeson ice, and the RNA quantity in each tube is then quantified with aspectrophotometer. For DNase treatment, 2 μg of RNA is diluted in 15 μLof RNase-free water. 1.75 μL of 10× DNase buffer and 0.75 μL of DNase isthen added, followed by incubation at 37° C. for 15 minutes. For reversetranscriptase treatment, 0.88 μL of EDTA (50 nM) is added, followed byincubation at 75° C. for 5 minutes. In a PCR tube, 0.5 μg ofDNase-treated RNA is mixed with 4 μL of iScript™ Reverse transcriptionSupermix (5×) and 20 μL of nuclease-free water. The mix is incubated ina PCR machine with the following program: 5 min at 25° C., 30 min at 42°C. and 5 min at 85° C. Newly synthesized cDNA is transferred in sterile1.5-mL tubes and diluted in 2 μL of nuclease-free water. 18 μL per wellof a qPCR machine (CFX-96™) mix is then added in a PCR plate foranalysis.

14.3 HOXB4-WT Transduction by His-CM18-PTD4 in THP-1 Cells: DoseResponses and Viability

THP-1 cells were cultured and tested in the protein transduction assayusing Protocol A as described in Example 9.1. Briefly, THP-1 cells wereplated at 30 000 cells/well one day before transduction. HOXB4-WTrecombinant protein (0.3, 0.9, or 1.5 μM; Example 14.1) was co-incubatedwith different concentrations of His-CM18-PTD4 (0, 0.5, 7.5, 0.8 or 1μM) and then exposed to THP-1 cells for 2.5 hours in the presence ofserum. The cells were subjected to real time-PCR analysis as describedin Example 14.2 to measure the mRNA levels of a target gene as a markerfor HOXB4 activity, which was then normalized to the target gene mRNAlevels detected in the negative control cells (no treatment), to obtaina “Fold over control” value. Total RNA levels (ng/μL) were also measuredas a marker for cell viability. Results are shown in Table 14.1 and FIG.42.

TABLE 14.1 Data from FIG. 42 Fold over Total RNA Cargo/ Conc. of Conc.of control in ng/μL shuttle agent shuttle HOXB4- (mean ± (mean ± (FIG.41) Cells (μM) WT (μM) St. Dev) St. Dev) No treatment THP-1 0 0   1 ±0.1 263 ± 0.4 (“Ø”) HOXB4-WT THP-1 0 1.5 4.3 ± 0.1 271 ± 6.0 alone(“TF”) His-CM18- THP-1 1 0 2.7 ± 0.3  252 ± 10.7 PTD4 alone (“FS”)His-CM18- THP-1 0.5 0.3 2.7 ± 0.6 255 ± 3.9 PTD4 + HOXB4- 0.9 4.3 ± 2.1 239 ± 17.5 WT 1.5 3.8 ± 0.7 269 ± 6.4 His-CM18- THP-1 0.75 0.3 4.2 ±1.2 248 ± 28  PTD4 + HOXB4- 0.9 5.7 ± 2.5 245 ± 31  WT 1.5 7.5 ± 2.8 230± 3.3 His-CM18- THP-1 0.8 0.3 9.1 ± 2.7 274 ± 4.4 PTD4 + HOXB4- 0.9 16.4± 1.7   272 ± 12.5 WT 1.5 22.7 ± 3.2  282 ± 4.7 His-CM18- THP-1 0.9 0.310.2 ± 2.5   280 ± 11.3 PTD4 + HOXB4- 0.9 18.7 ± 3.1  281 ± 9.2 WT 1.526.1 ± 3.5  253 ± 7.1 His-CM18- THP-1 1 0.3 10.5 ± 0.7   184 ± 12.3PTD4 + HOXB4- 0.9  17 ± 3.7  168 ± 16.2 WT 1.5 24.5 ± 3.9  154 ± 4.7

These results show that exposing THP-1 cells to a mixture of the shuttleagent His-CM18-PTD4 and the transcription factor HOXB4-WT for 2.5 hoursin the presence of serum results in a dose-dependent increase in mRNAtranscription of the target gene. These results suggest that HOXB4-WT issuccessfully delivered in an active form to the nucleus of THP-1 cells,where it can mediate transcriptional activation.

14.4 HOXB4-WT Transduction by His-CM18-PTD4 in THP-1 Cells: Time Courseand Viability (0 to 48 Hours)

THP-1 cells were cultured and tested in the protein transduction assayusing Protocol A as described in Example 9.1. Briefly, THP-1 cells wereplated at 30 000 cells/well one day before the first time courseexperiment. HOXB4-WT recombinant protein (1.5 μM; Example 14.1) wasco-incubated with His-CM18-PTD4 (0.8 μM) and then exposed to THP-1 cellsfor 0, 2.5, 4, 24 or 48 hours in presence of serum. The cells weresubjected to real time-PCR analysis as described in Example 14.2 tomeasure mRNA levels of a target gene as a marker for HOXB4 activity,which was then normalized to the target gene mRNA levels detected in thenegative control cells (no treatment), to obtain a “Fold over control”value. Total RNA levels (ng/μL) were also measured as a marker for cellviability. Results are shown in Table 14.2 and FIG. 43.

TABLE 14.2 Data from FIG. 43 Conc. of Conc. of Exposure Fold over TotalRNA in Cargo/shuttle shuttle HOXB4- time control (mean ± ng/μL (mean ±agent (FIG. 43) Cells (μM) WT (μM) (hours) St. Dev) St. Dev) Notreatment THP-1 0 0 —   1 ± 0.1 180 ± 0.4  (“Ctrl”) HOXB4-WT alone THP-10 1.5 2.5 h 3.4 ± 0.3 129 ± 10.7 (“TF”) His-CM18-PTD4 THP-1 0.8 0 2.5 h 1.2 ± 0.14 184 ± 6.0  alone (“FS”) His-CM18-PTD4 + THP-1 0.8 1.5  48 h0.27 ± 0.1   58 ± 11.2 HOXBA-WT  24 h  0.8 ± 0.14 74 ± 9.2   4 h 5.6 ±1.2 94 ± 7.1 2.5 h 9.1 ± 1.2 146 ± 11.6 0 3.9 ± 0.4 167 ± 13  

14.5 HOXB4-WT Transduction by His-CM18-PTD4 in THP-1 Cells: Time Courseand Viability (0 to 4 Hours)

THP-1 cells were cultured and tested in the protein transduction assayusing Protocol A as described in Example 9.1. Briefly, THP-1 cells wereplated at 30 000 cells/well one day before the first time courseexperiment. HOXB4-WT recombinant protein (0.3 μM; Example 14.1) wasco-incubated with His-CM18-PTD4 (0.8 μM) and then exposed to THP-1 cellsfor 0, 0.5, 1, 2, 2.5, 3 or 4 hours in presence of serum. The cells weresubjected to real time-PCR analysis as described in Example 14.2 tomeasure mRNA levels of a target gene as a marker for HOXB4 activity,which was then normalized to target gene mRNA levels detected in thenegative control cells (no treatment), to obtain a “Fold over control”value. Total RNA levels (ng/μL) were also measured as a marker for cellviability. Results are shown in Table 14.3 and FIG. 44.

TABLE 14.3 Data from FIG. 44 Conc. of Conc. of Exposure Fold over TotalRNA in Cargo/shuttle shuttle HOXB4- time control (mean ± ng/μL (mean ±agent (FIG. 42) Cells (μM) WT (μM) (hours) St. Dev) St. Dev) Notreatment THP-1 0 0 —   1 ± 0.1 289 ± 9.2  (“Ctrl”) His-CM18-PTD4 THP-10 0.3 2.5 h 2.5 ± 0.2 260 ± 7.1  alone (“FS”) HOXB4-WT alone THP-1 0.8 02.5 h   1 ± 0.14 264 ± 12.3 (“TF”) His-CM18-PTD4 + THP-1 0.8 0.3   4 h1.2 ± 0.1 198 ± 6.0  HOXBA-WT   3 h  1.3 ± 0.21 268 ± 12.5 2.5 h   2 ±0.3 275 ± 4.7    2 h 2.2 ± 0.2 269 ± 12.5 1 9.7 ± 2.6 268 ± 3.9  0.523.1 ± 2.0  266 ± 17.5 0   4 ± 0.5 217 ± 6.4 

14.6 HOXB4-WT Transduction by His-CM18-PTD4 in HeLa Cells:Immuno-Labelling and Visualization by Microscopy

Recombinant HOXB4-WT transcription factor (25 μM; Example 14.1) wasco-incubated with 35 μM of His-CM18-PTD4 and exposed to HeLa cells for10 seconds using Protocol B as described in Example 9.1. After a30-minute incubation to allow transduced HOXB4-WT to accumulate in thenucleus, the cells were fixed, permeabilized and immuno-labelled asdescribed in Example 3.2a. HOXB4-WT was labelled using a primary mouseanti-HOXB4 monoclonal antibody (Novus Bio # NBP2-37257) diluted 1/500,and a secondary anti-mouse antibody Alexa™-594 (Abcam #150116) diluted1/1000. Nuclei were labelled with DAPI. The cells were visualized bybright field and fluorescence microscopy at 20× and 40× magnificationsas described in Example 3.2, and sample results are shown in FIG. 45.Co-localization was observed between nuclei labelling (FIGS. 45A and45C) and HOXB4-WT labelling (FIGS. 45B and 45D), indicating thatHOXB4-WT was successfully delivered to the nucleus after 30 min in thepresence of the shuttle agent His-CM18-PTD4. White triangle windows showexamples of areas of co-localization between the nuclei (DAPI) andHOXB4-WT immuno-labels.

14.7 HOXB4-WT Transduction by Different Shuttle Agents in THP-1 Cells:Dose Responses and Viability

THP-1 cells were cultured and tested in the protein transduction assayusing Protocol A as described in Example 9.1. Briefly, THP-1 cells wereplated at 30 000 cells/well one day before the first time courseexperiment. HOXB4-WT recombinant protein (1.5 μM; Example 14.1)co-incubated with the shuttle agents His-CM18-PTD4, TAT-KALA, EB1-PTD4,His-C(LLKK)3C-PTD4 and His-CM18-PTD4-His at 0.8 μM, and then exposed toTHP-1 cells for 2.5 hours in presence of serum. The cells were subjectedto real time-PCR analysis as described in Example 14.2 to measure mRNAlevels of a target gene as a marker for HOXB4 activity, which was thennormalized to target gene mRNA levels detected in the negative controlcells (no treatment), to obtain a “Fold over control” value. Total RNAlevels (ng/μL) were also measured as a marker for cell viability.Results are shown in Table 14.4 and FIG. 47.

TABLE 14.4 Data from FIG. 47 Shuttle HOXB4-WT Fold over Total RNA Cargo/conc. Conc. Exposure control in ng/μL shuttle agent (μM) (μM) time (mean± St. Dev) (mean ± St. Dev) No treatment (“Ctrl”) 0 0 —   1 ± 0.09 240.3± 8.9  His-CM18-PTD4 0 1.5 2.5 h 2.5 ± 0.3 303.9 ± 7.6  alone (“FS”)HOXB4-WT alone 0.8 0 2.5 h   1 ± 0.11 251.9 ± 11.9 (“TF”)His-CM18-PTD4 + 0.8 1.5 2.5 h 44.5 ± 0.09   182 ± 5.97 HOXB4-WTTAT-KALA +  5.1 ± 0.21 222.4 ± 12.5 HOXB4-WT EB1-PTD4 + 6.4 ± 0.3 240.4± 4.71 HOXB4-WT His-C(LLKK)3C-  9.8 ± 0.19  175.3 ± 11.25 PTD4 +HOXB4-WT His-CM18-PTD4-His + 28.1 ± 2.61  91.4 ± 3.92 HOXB4-WT

Example 15 In Vivo GFP-NLS Delivery in Rat Parietal Cortex byHis-CM18-PTD4

The ability of the shuttle agent His-CM18-PTD4 to deliver GFP-NLS invivo in the nuclei of rat brain cells was tested.

In separate sterile 1.5-mL tubes, shuttle agent His-CM18-PTD4 wasdiluted in sterile distilled water at room temperature. GFP-NLS, used ascargo protein, was then added to the shuttle agent and, if necessary,sterile PBS was added to obtain the desired concentrations of shuttleagent and cargo in a sufficient final volume for injection in rat brain(e.g., 5 μL per each injection brain site). The shuttle agent/cargomixture was then immediately used for experiments. One negative controlwas included for the experiment, which corresponds to the injection ofthe GFP-NLS alone.

Bilateral injections were performed in the parietal cortex of threerats. In the left parietal cortex (ipsilateral), a mix composed of theshuttle agent (20 μM) and the GFP-NLS (20 μM) was injected, and in theright parietal cortex (contralateral), only the GFP-NLS (20 μM) wasinjected as a negative control. For surgical procedures, mice wereanesthetized with isoflurane. Then the animal was placed in astereotaxic frame, and the skull surface was exposed. Two holes weredrilled at the appropriate sites to allow bilateral infusion of theshuttle/cargo mix or GFP-NLS alone (20 μM) with 5-μL Hamilton syringe.Antero-posterior (AP), lateral (L), and dorso-ventral (DV) coordinateswere taken relative to the bregma: (a) AP 40.48 mm, L±3 mm, V−5 mm; (b)AP−2 mm, L±1.3 mm, V−1.5 mm; (c) AP−2.6 mm, L±1.5 mm, V−1.5 mm. Theinfused volume of the shuttle/cargo mix or cargo alone was 5 μL perinjection site and the injection was performed for 10 minutes. Afterthat, experimenter waited 1 min before removing the needle from thebrain. All measures were taken before, during, and after surgery tominimize animal pain and discomfort. Animals were sacrificed byperfusion with paraformaldehyde (4%) 2 h after surgery, and brain werecollected and prepared for microscopy analysis. Experimental procedureswere approved by the Animal Care Committee in line with guidelines fromthe Canadian Council on Animal Care.

Dorso-ventral rat brain slices were collected and analysed byfluorescence microscopy and results are shown in at 4× (FIG. 48A), 10×(FIG. 48C) and 20× (FIG. 48D) magnifications. The injection site islocated in the deepest layers of the parietal cortex (PCx). In thepresence of the His-CM18-PTD4 shuttle, the GFP-NLS diffused in cellnuclei of the PCx, of the Corpus Callus (Cc) and of the striatum (Str)(White curves mean limitations between brains structures). FIG. 48Bshows the stereotaxic coordinates of the injection site (black arrows)from the rat brain atlas of Franklin and Paxinos. The injection ofGFP-NLS in presence of His-CM18-PTD4 was performed on the left part ofthe brain, and the negative control (an injection of GFP-NLS alone), wasdone on the contralateral site. The black circle and connected blacklines in FIG. 48B show the areas observed in the fluorescent pictures(FIGS. 48A, 48C and 48D).

This experiment demonstrated the cell delivery of the cargo GFP-NLSafter its stereotaxic injection in the rat parietal cortex in thepresence of the shuttle agent His-CM18-PTD4. Results show the deliveryof the GFP-NLS in the nucleus of cells from the deeper layers of theparietal cortex (injection site) to the corpus callus and the dorsallevel of the striatum (putamen). In contrast, the negative control inwhich GFP-NLS is only detectable locally around the injection site. Thisexperiment shows that shuttle agent induced nuclear delivery of thecargo in the injection site (parietal cortex) and its diffusion throughboth neighboring brain areas (corpus callus and striatum rat brain).

Example A Physiochemical Properties of Domain-Based Peptide ShuttleAgents

A plurality of different peptides was initially screened with the goalof identifying polypeptide-based shuttle agents that can deliverindependent polypeptide cargos intracellularly to the cytosol/nucleus ofeukaryotic cells. On one hand, these large-scale screening efforts ledto the discovery that domain-based peptide shuttle agents (see Examples1-15), comprising an endosome leakage domain (ELD) operably linked to acell penetrating domain (CPD), and optionally one or more histidine-richdomains, can increase the transduction efficiency of an independentpolypeptide cargo in eukaryotic cells, such that the cargo gains accessto the cytosol/nuclear compartment Conversely, these screening effortsrevealed some peptides having no or low polypeptide cargo transductionpower, excessive toxicity, and/or other undesirable properties (e.g.,poor solubility and/or stability).

Based on these empirical data, the physiochemical properties ofsuccessful, less successful, and failed peptides were compared in orderto better understand properties common to the more successful shuttleagents. This approach involved manually stratifying the differentpeptides according to transduction performance with due considerationto, for example: (1) their solubility/stability/ease of synthesis; (2)their ability to facilitate endosomal escape of calcein (e.g., seeExample 2); (3) their ability to deliver one or more types ofindependent polypeptide cargo intracellularly, as evaluated by flowcytometry (e.g., see Examples 3-6 and 8-15) in different types of cellsand cell lines (e.g., primary, immortalized, adherent, suspension, etc.)as well as under different transduction protocols; and (4) their abilityto deliver polypeptide cargos to the cytosol and/or nucleus, asevaluated by fluorescence microscopy (e.g., for fluorescently labelledcargos), increased transcriptional activity (e.g., for transcriptionfactor cargos), or genome editing capabilities (e.g., for nucleasecargos such as CRISPR/Cas9 or CRISPR/Cpf1) (e.g., see Examples 3-6 and8-15), and toxicity towards different types of cells and cell lines(e.g., primary, immortalized, adherent, suspension, etc.), underdifferent transduction protocols.

In parallel to the above-mentioned manual curation, the transductionpower and cellular toxicity of each peptide for a givenfluorescently-labelled cargo (GFP, GFP-NLS, or fluorescently-labelledantibodies) and cell line were combined into a single “transductionscore” as a further screening tool, which was calculated as follows:[(Highest percentage transduction efficiency observed by flow cytometryfor a given peptide in a cell type)×(Percentage viability for thepeptide in the tested cell line)]/1000, giving an overall transductionscore between 0 and 10 for a given cell type and polypeptide cargo.These analyses identified domain-based peptides having transductionscores ranging from about 8 (e.g., for successful domain-based peptideshuttle agents) to as low as 0.067 (e.g., for single-domain negativecontrol peptides).

The above-mentioned manual curation and “transduction score”-basedanalyses revealed a number of parameters that are common to manysuccessful domain-based shuttle agents. Some of these parameters arelisted in the Table A1. An example of a “transduction score”-basedanalyses in HeLa cells using GFP as a polypeptide cargo is shown inTable A2. Other transduction score-based analyses using cell lines otherthan HeLa and polypeptides cargos other than GFP, were also performedbut are not shown here for brevity.

No successful shuttle agents were found having less than 20 amino acidresidues in length (see parameter 1 in Tables A1 and A2). The four aminoacids alanine, leucine, lysine and arginine, were the principal and mostrecurrent residues in most of the successful shuttle agents (35-85% ofresidues of the peptide; see parameter 10). These residues dictate thealpha-helical structure and amphiphilic nature of these peptidesequences (parameters 2-5). There was often a balance between thepercentages of A/L residues (15-45%) and K/R residues (20-45%) in theshuttle agents (parameters 11, 12 and 14), and the percentages ofnegatively charged residues was often found to be not greater than 10%(parameter 14). Conversely, the sixteen other amino acid residues (otherthan A, L, K, and R) represented generally between 10-45% of the shuttleagents (parameter 15). Successful shuttle agents generally had apredicted isoelectric point (pI) of between 8-13 (parameter 7), and apredicted net charge greater than or equal to +4 (parameter 6), withdCM18-TAT-Cys having a predicted net charge of as high as +26.Hydrophobic residues (A, C, G, I, L, M, F, P, W, Y, V) composedgenerally between 35-65% of the shuttle agents, and neutral hydrophilicresidues (N, Q, S, T) represented generally from 0-30% (parameters 8 and9).

As shown in Table A2, the most successful shuttle agents (e.g.,transduction scores above 5.0) generally had few parameters outside theranges set forth in Table A1. However, significant increases intransduction efficiency were also observed for shuttle agents in whichseveral parameters were not satisfied, depending for example on theextent to which the unsatisfied parameters fall outside the recommendedrange, and/or on whether other parameters fall closer to the middle of arecommended range. Thus, shuttle agents having several parameters whichfall within “optimal” ranges may compensate for other parameters fallingoutside of the recommended ranges. As mentioned above, peptides shorterthan 20 amino acids did not show any significant transduction ability(e.g. transduction scores less than 0.4), regardless of how many otherparameters were satisfied. Among the peptides greater than 20 aminoacids in length and having transduction scores lower than 0.4, VSVG-PTD4(score of 0.35) failed to satisfy six parameters, while JST-PTD4 (scoreof 0.083) failed to satisfy ten parameters. KALA (score of 0.12) failedto satisfy four parameters, with parameters 11 and 14 far exceeding therecommended ranges, reflecting an overabundance of A/L residues and alarge imbalance between the percentages of A/L and L/R residues. It isto be understood that the transduction score ranges appearing the TableA2 are arbitrarily selected, and that other ranges can be selected andare within the scope of the present description.

TABLE A1 General physicochemical properties of successful domain-basedpeptide shuttle agents Parameter Description Result 1 Minimum length Theminimum length of peptide shuttle agent. 20 amino acids 2 AmphipathicPeptide shuttle agent comprises a predicted amphipathic alpha-helixconformation, (Based on 3D Yes alpha-helix modeling using PEP-FOLD, anonline resource for de novo peptide structure prediction:http://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD/). 3Positively-charged Predicted amphipathic alpha-helix conformationcomprises a positively-charged hydrophilic face rich Yes surface in Rand/or K residues, (Based on observation of at least 3 K/R residuesclustered to one side of a helical wheel modeling, using the onlinehelical wheel projection tool available at:http://rzlab.ucr.edu/scripts/wheel/wheel.cgi). 4 % Highly Predictedamphipathic alpha-helix conformation comprises a highly hydrophobic corecomposed of  12-50% hydrophobic core spatially adjacent L, I, F, V, W,and/or M residues representing a percentage of the overall peptidesequence (calculated excluding histidine-rich domains). This parameterwas calculated by first arranging the amino acids of the peptide in anopened cylindrical representation, then delineating an area ofcontiguous highly hydrophobic residues (L, I, F, V, W, M), as shown inFIG. 49A, right panel. The number of highly hydrophobic residuescomprised in this delineated highly hydrophobic core was then divided bythe total amino acid length of the peptide, excluding N- and/orC-terminal histidine-rich domains. For example, for the peptide shown inFIG. 49A, there are 8 residues in the delineated highly hydrophobiccore, and 25 total residues in the peptide (excluding the terminal 12histidines). Thus, the highly hydrophobic core is 32% (8/25). 5Hydrophobic Calculated hydrophobic moment (μ; calculated while excludinghistidine-rich domains), using the 3.5-11 moment online helical wheelprojection program available from:http://rzlab.ucr.edu/scripts/wheel/wheel.cgi. 6 Net charge Predicted netcharge at physiological pH (calculated from the side chains of K, R, D,and E residues), ≥+4 7 pI Predicted isoelectric point (pI). (Calculatedwith the Prot Param software available at:   8-13http://web.expasy.org/protparam/). 8 % hydrophobic Overall percentage ofhydrophobic residues (A, C, G, I, L, M, F, P, W, Y, V) in the peptideshuttle  35-65% residues agent. 9 % neutral Overall percentage ofneutral hydrophilic residues (N, Q, S, T) in the peptide shuttle agent.  0-30% hydrophilic residues 10 % A, L, K, R Overall percentage ofresidues in the peptide shuttle agent which are A, L, K, or R.  35-85%11 % A or L Overall percentage of residues in the peptide shuttle agentwhich are A or L. (Number of A + L  15-45% residues)/(Total number ofresidues), with there being at least 5% of L in the peptide. 12 %Positive Overall percentage of residues in the peptide shuttle agentwhich are K or R. (Number of K + R  20-45% residues residues)/(Totalnumber of residues). 13 % Negative Overall percentage of residues in thepeptide shuttle agent which are D or E. (Number of D + E   0-10%residues residues)/(Total number of residues). 14 Difference Overalldifference (absolute value) between the percentage of A or L (parameter11) and the ≤10% between % of A/L percentage of K or R (parameter 12) inthe peptide shuttle agent. (Parameter 11) − (Parameter 12). and K/R 15 %infrequent Overall percentage of residues which are Q, Y, W, P, I, S, G,V, F, E, D, C, M, N, T or H (i.e., not A,  10-45% amino acid L, K, orR). (Number of Q + Y + W + P + I + S + G + V + F + E + D + C + M + N +T + H residues residues)/(Total number of residues).

Example B Rational Design of Synthetic Peptide Shuttle Agents

The parameters set forth in Table A1, and empirical knowledge gained(e.g., from Examples 1-15), were used to manually design the peptideslisted in Table B1 in order to evaluate whether the parameters can beused for designing successful peptide shuttle agents.

The peptides listed in Table B1 were tested for their ability totransduce GFP-NLS cargo (see Example 3.4) in HeLa cells, using theprotein transduction assay as generally described in Example 3.1a.GFP-NLS recombinant protein (10 μM) was co-incubated with 10 μM of thepeptides and then exposed to HeLa cells for 1 min. The cells weresubjected to flow cytometry analysis as described in Example 3.3.Results are shown in Tables B2 and B3, A “transduction score”-basedanalysis was also performed as discussed is Example A, and the resultsare shown in Table B4. Successful nuclear delivery of the transducedGFP-NLS (generally after only 1 minute of exposure to the peptide) wasconfirmed by fluorescence microscopy as described in Example 3.2 (datanot shown).

Peptides FSD1-FSD5 were initially designed based on the successfuldomain-based shuttle agent His-CM18-PTD4-His, with peptides FSD1-FSD4being designed to intentionally unrespect one or more parameters setforth in Table A1, and FSD5 being designed to respect all fifteenparameters. As can be seen from Table B2, peptides FSD1-FSD4 displayedtransduction efficiencies ranging from 2.45% to 37.6%. In contrast, thepeptide FSD5 displayed high transduction efficiency (70.5%) and lowtoxicity (cell viability of 86%).

3-dimensional modeling using PEP-FOLD, an online resource for de novopeptide structure prediction, predicted an alpha-helical conformationfor FSD5 (see FIG. 49C;http://bioserv.rpbs.univ-paris-diderot.fr/services/PEP-FOLD). Incontrast, the peptide VSVG-PTD4, which showed only 3.5% transductionefficiency (see Table 10.3a), was predicted to adopt a differentstructure which included a shorter alpha helix, short beta-sheets (whitearrows), and random coils (white shapeless lines).

Helical wheel projections and side opened cylindrical representations ofFSD5 and VSVG-PTD4 shown in FIGS. 49A and 49B (adapted from:http://rzlab.ucr.edu/scripts/wheel/wheel.cgi) illustrate the amphipathicnature of FSD5, as compared to VSVG-PTD4. The geometrical shape of eachamino acid residue corresponds to its biochemical property based on theside chain of the residue (i.e., hydrophobicity, charge, orhydrophilicity). One of the main differences between the two openedcylindrical representations of FSD5 and VSVG-PTD4 is the presence of ahighly hydrophobic core in FSD5 (outlined in FIG. 49A, left and rightpanels), which is not present in VSVG-PTD4. The cylinder in the lowermiddle panels of FIGS. 49A and 49B represent simplified versions of theopened cylindrical representations in the right panels, in which: “H”represents the high hydrophobic surface area; “h” represents lowhydrophobic surface area; “+” represents positively charged residues;and “h” represent hydrophilic residues.

In light of the high transduction efficiency of FSD5, we used thisshuttle agent as model to design peptides FSD6-FSD26. As shown in TableB2, a relatively high degree of amino acid substitutions was possiblewithout completely losing transduction power, provided that most of thedesign parameters set forth in Table A1 were respected. The only peptidethat displayed nearly a complete loss transduction efficiency amongFSD6-FSD26 was FSD6, which is not predicted to adopt an amphipathicalpha-helix structure. Interestingly, peptide FSD18 showed high toxicityin HeLa cells when used at 10 μM, but showed high transductionefficiency and relatively low toxicity when used in other types of cells(see Examples E and G), suggesting that peptide toxicity may varydepending on the type of cells. 3-dimensional modeling using PEP-FOLDpredicted two separate alpha-helices for FSD18 (see FIG. 49D).

Peptides FSN1-FSN8 were designed to explore the effects on transductionefficiency when one or more of the design parameters set forth in TableA1 are not respected.

TABLE B1 Manually-designed synthetic peptides and shuttle agents SEQPep- ID Length MW tide NO: Amino acid sequence (a.a.) (kDa) pI ChargeFSD1 104 HHHHHHKWKLLRRAAKKAARRYLKLLLKQLKLHHHHHH 38 4.85 12.03 11+/0−FSD2 105 HHHHHHWLKLLRRAAKKAARLYRKLLRKARKLHHHHHH 38 4.86 12.31 12+/0−FSD3 106 HHHHHHKRKKKRRAKKKRAWLYLALLLWALALHHHHHH 38 4.87 12.03 11+/0−FSD4 107 HHHHHHKRQLKRKLRKWKRLLRLLRLLARLWLHHHHHH 38 5.1 12.78 12+/0− FSD5108 HHHHHHLLKLWSRLLKLWTQGRRLKAKRAKAHHHHHH 37 4.68 12.49  9+/0− FSD6 109HHHHHHWYLALLALYWQRAKAKTRQRRRHHHHHH 34 4.49 11.84  7+/0− FSD7 110HHHHHHWARLARAFARAIKKLYARALRRQARTG 33 3.99 12.4  9+/0− FSD8 111HHHHHHKWKLARAFARAIKKLYARALRRQARTG 33 4.02 12.31 10+/0− FSD9 112HHHHHHKWKLARAFARAIKKLYARALRRQARTGHHHHHH 39 4.85 12.31 10+/0− FSD10 113KWKLARAFARAIKKLGGSGGGSYARALRRQARTG 34 3.66 12.31 10+/0− FSD11 114HHHHHHKWKLARAFARALRAIKKLYARALRRQARTG 36 4.36 12.4 11+/0− FSD12 115KWKLARAFARAIKKLYARALRRQARTG 27 3.2 12.31 10+/0− FSD13 116HHHHHHKWAKLLRAFAKAIKKLYARLARRQARTGHHHHHH 40 4.93 12.19 10+/0− FSD14 117HHHHHHLALARWARYFRILAKLKRTKRGQAKAHHHHHH 38 4.73 12.19  9+/0− FSD15 118HHHHHHKWKIARAFARSLKKLYARLLARQAKTGHHHHHH 39 4.79 12.02  9+/0− FSD16 119HHHHHHLLKLWSRLLKLWTQGRRLKAKRAKA 31 3.86 12.49  9+/0− FSD17 120HHHHHHLAKLFKWLRALIRQGAKRKTKRASAHHHHHH 37 4.56 12.49  9+/0− FSD18 121LLKLWSRLLKLWTQGGSGGGSGRRLKAKRAKA 32 3.49 12.49  9+/0− FSD19 122HHHHHHLLKLWSRLLKTWTQGRRLKAKSAQASTRQAHHHHHH 36 4.32 12.48  8+/0− FSD20123 HHHHAAVLKLWKRLLKLFRKGRRLKAKRAKAKR 33 4.12 12.71 14+/0− FSD21 124HHHHHHFLKIWSRLIKIWTQGRRKGAQAAFR 31 3.85 12.48  7+/0− FSD22 125HHHHHHVLKLWSRILKAFTQGRRMAAKRAKCNHHHHHH 32 3.87 12.02  8+/0− FSD23 126HHHHHHLLKLWSRLLKEWTQGRRLEAKRAEAHHHHHH 31 3.88 10.93  7+/3− FSD24 127HHHHHHLLCLWSRLLKLWTQGERLKAKCAKACER 34 4.14  9.75  7+/2− FSD25 128HHHHHHVWKLFWTLLAAIYGRGKARQKRAKRQARG 35 4.25 12.19  9+/0− FSD26 129ALLGLFIKWVKKVGTLFRKAKAGAQNRRAKAQKGK 35 3.88 12.33 11+/0− FSN1 130HHHHHHKRKRRSKARKLWTQGWLLLALLALAHHHHHH 31 3.86 12.49  9+/0− FSN2 131HHHHHHKLKLRSRLKWGRTQLWRALAKKALLHHHHHH 31 3.86 12.49  9+/0− FSN3 132HHHHHHQFLCFWLNKMGKHNTVWHGRHLKCHKRGKG 31 3.82 11.75  7+/0− FSN4 133HHHHHHLLYLWRRLLKFWCAGRRVYAKCAKAYGCF 35 4.23 10.06  7+/0− FSN5 134HHHHHHLLKLWRRLLKLFRKALRALAKRAKSALKRAQAA 39 4.68 12.71 12+/0− FSN6 135HHHHHHLLKLWSRLLKLWTQALRALAKRAKALAHHHHHH 33 3.96 12.31  7+/0− FSN7 136LIKLWSRFIKFWTQGRRIKAKLARAGQSWFG 31 3.75 12.48  8+/0− FSN8 137HHHHHHFRKLWLAIVRAKK 19 2.4 12.02  5+/0− FSD43 169[Refer to Sequence Listing] — — — — to to FSD116 242 Results computedusing ProtParam ™ online tool available from ExPASy ™ BioinformaticsResource Portal (http://web.expasy.org/protparam/); pI: isoelectricpoint; Charge: Total number of positively (+) and negatively (−) chargedresidues

TABLE B2 Transduction of GFP-NLS in HeLa cells Mean % Cell cells withviability GFP signal (%) (±St. Dev.; (±St Dev.; Cells Peptide n = 3) n =3) Design comments HeLa No  0.41 ± 0.015 100 n/a peptide FSD1  37.6 ±3.44 60.3 ± 6.18 Low % of infrequent amino acids (Q, Y, W, P, I, S, G,V, F, E, D, C, M, N, T, H) FSD2  11.9 ± 1.69 76.3 ± 5.99 Highhydrophobic moment FSD3  2.45 ± 0.32 91.1 ± 6.37 No predictedamphipathic alpha helix FSD4  6.60 ± 0.84 86.1 ± 8.15 Low % ofhydrophobic amino acids; Low % of infrequent amino acids (Q, Y, W, P, I,S, G, V, F, E, D, C, M, N, T, H) FSD5  70.5 ± 6.44   86 ± 7.45 — FSD6   1 ± 0.12 88.1 ± 7.66 No predicted amphipathic alpha helix FSD7 78.30± 5.11 38.5 ± 3.48 — FSD8 62.30 ± 5.61 64.8 ± 7.59 — FSD9 68.21 ± 6.3567.3 ± 5.19 — FSD10 73.23 ± 4.94 79.8 ± 4.73 — FSD11 68.29 ± 3.11 60.9 ±7.59 — FSD12 61.58 ± 5.33 67.8 ± 4.83 — FSD13 75.94 ± 7.48 49.5 ± 5.13High hydrophobic moment FSD14 43.25 ± 5.35 92.8 ± 7.42 — FSD15 54.97 ±4.28 96.1 ± 2.61 — FSD16 57.34 ± 4.11 88.2 ± 2.66 — FSD17 52.83 ± 6.6999.1 ± 2.09 — FSD18 77.11 ± 3.25 82.4 +/− 4.71 — FSD19 55.17 ± 4.62 80.6± 5.36 — FSD20 75.23 ± 5.91 65.4 ± 6.18 — FSD21 46.74 ± 4.03 75.6 ± 5.99— FSD22 45.09 ± 3.95 80.2 ± 7.21 — FSD23 50.34 ± 4.29 65.3 ± 5.44 —FSD24 37.48 ± 4.08 75.3 ± 3.93 — FSD25 32.67 ± 3.17 71.7 ± 5.08 — FSD2647.63 ± 4.19 59.26 ± 1.27  — “—” = No parameters outside the limits setforth in Table A1.

TABLE B3 Transduction of GFP-NLS in HeLa cells Mean % Cell cells withviability GFP signal (%) (±St Dev.; (±St. Dev.; Cells Peptide n = 3) n =3) Design comments HeLa No 0.38 ± 0.02 100 peptide FSN1 9.14 ± 0.93 94.3 ± 3.07 Low hydrophobic moment; weak amphiphilic structure FSN212.13 ± 2.06   91.3 ± 4.66 Weak hydrophobic surface FSN3  1.86 ± 97.15 97.2 ± 2.03 No predicted alpha-helical stricture FSN4 5.84 ± 0.49  90.5± 4.18 >65% hydrophobic amino acids FSN5 13.29 ± 1.24  85.36 ± 6.16Alanine + Leucine > 40% FSN6 15.74 ± 1.63  32.63 ± 4.26 High hydrophobicmoment; difference between A/L and K/R residues is > 20% FSN7 3.56 ±0.36 93.45 ± 3.61 No predicted alpha-helical structure; high hydrophobicmoment; 55% of infrequent residues (other than A, L, K, R) FSN8 3.52 ±0.41 94.53 ± 3.72 Peptide length is less than 20

The primary amino acid sequences of peptides FSD5, FSD16, FSD18, FSD19,FSD20, FSD22, and FSD23 are related, as shown in the alignment below.

FSD23

FSD19

FSD5

FSD18

FSD16

FSD20

FSD22

  

  (SEQ ID NO: 158)         (SEQ ID NO: 159)

Example C Computer-Assisted Design of Synthetic Peptide Shuttle AgentsC.1 Machine-Learning-Assisted Design Approach

The peptides listed in Table C1 were designed using an algorithmdescribed in an article by Sébastien Giguère et al. entitled “MachineLearning Assisted Design of Highly Active Peptides for Drug Discovery”(Giguère et al., 2014). This computational prediction method is foundedon the use of algorithms based on the Kernel and machine learningmethods (Shawe-Taylor J. and Cristianini N., 2004). These algorithms aimto sort peptides with maximal bioactivity depending on a biologicaleffect of interest. Here, we considered all the peptides that we testedto date in protein transduction assays, and separated them into threedistinct groups. The composition of the groups was based on a“transduction score” calculated as described in Example A. Group 1 wascomposed of peptides demonstrating efficient cell delivery with lowtoxicity; Group 2 was composed of peptides demonstrating efficient celldelivery but with elevated toxicity; and Group 3 was composed ofpeptides that did not demonstrate any significant polypeptide cargotransduction ability.

The scores of the peptides in each group were used as starting datapoints for the generation of further peptide variants. The algorithm wasprogrammed to use the peptide sequences and the scores of the peptidesof Group 1 as the positive references for the prediction of peptidevariants with efficient transduction ability. The sequences and thescores of Groups 2 and 3 were included as negative controls in thealgorithm to delineate the search field. The peptide variants generatedby the algorithm were limited to those having a length of 35 aminoacids. After running, the prediction method generated sixteen sequences(FSD27 to FSD42). After analyzing the sequences of these sixteensequences with respect to the design parameters set forth in Table A1,only peptides FSD27, FSD34 and FSD40 satisfied all of the designparameters (see Table C2). The other peptide variants had one or moreparameters outside those set forth in Table A1.

TABLE C1 Machine-designed synthetic peptides and shuttle agents testedSEQ Pep- ID Length MW tide NO: Amino acid sequence (aa) (kDa) pI ChargeFSD27 138 HHHHHHKWKLFWEAKLAKYARAAARQARAARQARA 35 4.21 11.85  9+/1− FSD28139 HHHHHHHMAHLWESNARKFWKKAFAQHAAAHIAEA 35 4.18  9.7  4+/2− FSD29 140LHHHSHHLIHIWLLFKLKLKKKKAARRARRARRHH 35 4.43 12.71 12+/0− FSD30 141HHHHHHCLLKKWEAKLAKKIGGGGRQARAKALAKA 35 3.88 10.74  9+/1− FSD31 142YHHHHHKWKKRWEAKLAKALRAAGRQARAKALAKA 35 4.12 11.62 11+/1− FSD32 143IVRHEHCMIHLWYKNLAKYCSTSHARRLARRRAHH 35 4.35 10.92  8+/1− FSD33 144HHHHHHHHRQRRRWEARGGFLGGGGYARAARQARA 35 4.12 12.22  8+/1− FSD34 145HHHHHHKLIHIWEAKLFKKIRAAARQARARRAAKA 35 4.19 12.19 10+/1− FSD35 146HHHHHHKLLKRWEAKLAKALAKALAKHLAKALAKA 35 3.97 10.82  9+/1− FSD36 147HHHHHHCLIHIWEAKLAKKCGGGGYARAAARQARA 35 3.89 10.06  6+/1− FSD37 148RLHHSHHLIHIWLLFKLKLKKKKRAARRARRHHHL 35 4.47 12.71 12+/0− FSD38 149GHHHHHHHLIHIWEAKLAKLAKALARAAARQARAK 35 3.99 11.74  7+/1− FSD39 150HHHHHHHHRQRRRWEARGFLGGGGYARAARQARAA 35 4.14 12.22  8+/1− FSD40 151YGRKKRYMLRLWYQNLRMYCKKAYAQHRARQHAKL 35 4.53 10.81 11+/0− FSD41 152LHHHHHKLIHIWEAKLAKALAKALARRAAARQARA 35 3.99 12.02  8+/1− FSD42 153HHHHHHCMKVVWEIVLAKYKGGGGRARAASRRARA 35 3.98 11.47  8+/1− Resultscomputed Lising ProtParam ™ online tool available from ExPASy™ Bioinformatics Resource Portal (http://web.expasy.org/protparam/); pI:Isoelectric point; Charge: Total number of positively (+) and negatvely(−) charged residues

TABLE C2 FSD27 to FSD42 sequences and properties Comments concerning outof limit Peptide parameter(s) with respect to Table A1 FSD27 No out oflimit parameters. FSD28 No predicted amphiphilic alpha-helical structureHighly hydrophobic core <12% of the total surface Net charge below +4(+2) Low percentage K/R residues (11.5%) Difference between % A/L and %K/R greater than 10% (17.1%) FSD29 No predicted amphiphilicalpha-helical structure Highly hydrophobic core <12% of the totalsurface Low hydrophobic moment (3.3) FSD30 Highly hydrophobic core <12%of the total surface Low hydrophobic moment (2.6) FSD31 Highlyhydrophobic Core <12% of the total surface FSD32 No predictedamphiphilic alpha-helical structure Highly hydrophobic core <12% of thetotal surface FSD33 Highly hydrophobic core <12% of the total surfaceLow hydrophobic moment (2.1) Less than 5% leucines (2.9%) Differencebetween % A/L and % K/R greater than 10% (22.9%) FSD34 No out of limitparameters. FSD35 Highly hydrophobic core <12% of the total surface Lowhydrophobic moment (2.8) High % of A/L (48.6%) Difference between % A/Land % K/R greater than 10% (22.8%) Low % of total non-A/L/K/R residues(2.85%) FSD36 No predicted amphiphilic alpha-helical structure Lowhydrophobic moment (3.3) Highly hydrophobic Core <12% of the totalsurface Difference between % A/L and % K/R greater than 10% (11.4%)FSD37 No predicted amphiphilic alpha-helical structure FSD38 Differencebetween % A/L and % K/R greater than 10% (20%) FSD39 Highly hydrophobiccore <12% of the total surface FSD40 No out of limit parameters. FSD41High % of A/L (46%) Difference between % A/L and % K/R greater than 10%(23%) FSD42 Less than 5% leucines (2.9%)

HeLa cells were cultured and tested in the protein transduction assaydescribed in Example 3.1a. GFP-NLS recombinant protein (10 μM) wasco-incubated with 10 μM of the peptide and then exposed to HeLa cellsfor 1 min. The cells were subjected to flow cytometry analysis asdescribed in Example 3.3. Results are shown in Table C3.

TABLE C3 Transduction of GFP-NLS in HeLa cells by machine-designedsynthetic peptides Mean % cells with GFP signal Cell viability (%) CellsPeptide (±St. Dev.; n = 3) (±St. Dev.; n = 3) HeLa No peptide 0.22 ±0.03 100 FSD27* 25.49 ± 6.52  96.6 ± 4.94 FSD28 0.83 ± 0.29 99.4 ± 4.04FSD29 6.43 ± 2.6  89.8 ± 8.48 FSD30 1.75 ± 1.14 99.1 ± 0.52 FSD31 6.90 ±1.27 97.8 ± 4.22 FSD32 3.12 ± 1.03 99.2 ± 3.37 FSD33 0.68 ± 0.45 98.1 ±1.73 FSD34* 32.89 ± 8.9  97.9 ± 8.18 FSD35 2.08 ± 0.92 81.7 ± 3.45 FSD360.35 ± 0.2  98.9 ± 0.38 FSD37* 11.57 ± 2.99  73.9 ± 2.62 FSD38 4.61 ±1.33 98.1 ± 7.35 FSD39 0.23 ± 0.09 97.3 ± 2.07 FSD40* 32.66 ± 0.77  83.9± 4.16 FSD41* 36.99 ± 0.88  79.5 ± 0.33 FSD42 1.59 ± 0.39 97.3 ± 1.07*Peptides demonstrating transduction efficiencies above 10% appear inbold.

Interestingly, the three peptides generated using the algorithm thatrespected all of the design parameters set forth in Table A1 (i.e.,FSD27, FSD34 and FSD40) each demonstrated 25-33% transductionefficiency, with cell viabilities ranging from 83.9%-98%. The otherpeptides generally demonstrated transduction efficiencies below 12%,except for FSD41, which demonstrated a transduction efficiency of 37%(albeit with higher toxicity than FSD27, FSD34, and FSD40). Althoughonly a single parameter (i.e., efficiency score) was used to program thealgorithm, the results with FSD27, FSD34 and FSD40 validate theusefulness of the design parameters set forth in Table A1.

C.2 Computer-Assisted Generation of Peptide Variants

A computer-assisted design approach was employed to demonstrate thefeasibility of designing and generating peptide variants that respectmost or all of the design parameters set forth in Table A1. First, thisapproach involved manually considering and comparing the primary aminoacid sequences of structurally different yet successful peptide shuttleagents to identify general consensus sequences that lead to structuralparameters (2), (3) and (4) being respected (i.e., amphipathicalpha-helix formation, a positively-charged face, and a highlyhydrophobic core of 12%-50%). Second, the approach involvedcomputer-assisted random peptide sequence generation followed bydescriptors filtering, implementing the consensus sequences, and one ormore of the design parameters (1) and (5)-(15), in order to generate alist of peptide variants that respect nearly all of the designparameters. This is discussed in more detail below.

First, the primary amino acid sequences of peptides shown herein to haverelatively high transduction efficiency scores were compared usingonline multiple sequence alignment tools, including CLUSTALW 2.1(http://www.genome.jpltools-bin/clustalw); MUltiple Sequence Comparisonby Log-Expectation (MUSCLE) (https://www.ebi.ac.uk/Tools/msa/muscle/);and PRALINE (http://www.ibi.vu.nl/programs/pralinewww/). The peptidesselected for comparison included the following eleven peptides:His-CM18-PTD4; EB1-PTD4; His-C(LLKK)₃C-PTD4; FSD5; FSD10; FSD19; FSD20;FSD21; FSD44; FSD46; and FSD63, but the analyses were not limited toonly these eleven peptides. FIG. 49G shows an alignment of the elevenexemplary peptides using PRALINE, wherein the “Consistency” scores atthe bottom of each aligned residue position represents the degree ofconservation at that residue position (zero being the least conserved,and ten being the most conserved). For example, the alanine (A) atrelative position 29 was conserved in all eleven peptides shown in FIG.49G, and this was assigned a “Consistency” score of 10. Such multiplesequence analyses of the library of described peptides herein (andothers) revealed the following general structures:

(a) [X1]-[X2]-[linker]-[X3]-[X4]  (Formula 1);

(b) [X1]-[X2]-[linker]-[X4]-[X3]  (Formula 2);

(c) [X2]-[X1]-[linker]-[X3]-[X4]  (Formula 3);

(d) [X2]-[X1]-[linker]-[X4]-[X3]  (Formula 4);

(e) [X3]-[X4]-[linker]-[X1]-[X2]  (Formula 5);

(f) [X3]-[X4]-[linker]-[X2]-[X1]  (Formula 6);

(g) [X4]-[X3]-[linker]-[X1]-[X2]  (Formula 7); or

(h) [X4]-[X3]-[linker]-[X2]-[X1]  (Formula 8),

where [X1], [X2], [X3], [X4], and [linker] are as defined in the tablebelow:

TABLE C4 [X1] 2[ϕ]-1[+]-2[ϕ]-1[ζ]-1[+]- or 2[ϕ]-1[+]-2[ϕ]-2[+]- or1[+]-1[ϕ]-1[+]-2[ϕ]-1[ζ]-1[+]- or 1[+]-1[ϕ]-1[+]-2[ϕ]-2[+]- [X2]-2[ϕ]-1[+]-2[ϕ]-2[ζ]- or -2[ϕ]-1[+]-2[ϕ]-2[+]- or-2[ϕ]-1[+]-2[ϕ]-1[+]-1[ζ]- or -2[ϕ]-1[+]-2[ϕ]-1[ζ]-1[+]- or-2[ϕ]-2[+]-1[ϕ]-2[+]- or -2[ϕ]-2[+]-1[ϕ]-2[ζ]- or-2[ϕ]-2[+]-1[ϕ]-1[+]-1[ζ]- or -2[ϕ]-2[+]-1[ϕ]-1[ζ]-1[+]- [linker] -Gn-or -Sn- or -(GnSn)n- or -(GnSn)nGn- or -(GnSn)nSn- or -(GnSn)nGn(GnSn)n-or -(GnSn)nSn(GnSn)n- [X3] -4[+]-A- or -3[+]-G-A- or -3[+]-A-A- or-2[+]-1[ϕ]-1 [+]-A- or -2[+]-1[ϕ]-G-A- or -2[+]-1[ϕ]-A-A- or-2[+]-A-1[+]-A or -2[+]-A-G-A or -2[+]-A-A-A- or -1[ϕ]-3[+]-A- or-1[ϕ]-2[+]-G-A- or -1[ϕ]-2[+]-A-A- or -1[ϕ]-1[+]-1[ϕ]-1[+]-A or-1[ϕ]-1[+]-1[ϕ]-G-A or -1[ϕ]-1[+]-1[ϕ]-A-A or -1[ϕ]-1[+]-A-1[+]-A or-1[ϕ]-1[+]-A-G-A or -1[ϕ]-1[+]-A-A-A or -A-1[+]-A-1[+]-A or-A-1[+]-A-G-A or -A-1[+]-A-A-A [X4] -1[ζ]-2A-1[+]-A or -1[ζ]-2A-2[+] or-1[+]-2A-1[+]-A or -1[ζ]-2A-1[+]-1[ζ]-A-1[+] or -[ζ]-A-1[ζ]-A-1[+] or-2[+]-A-2[+] or -2[+]-A-1[+]-A or -2[+]-A-1[+]-1[ζ]-A-1[+] or-2[+]-1[ζ]-A-1[+] or -1[+]-1[ζ]-A-1[+]-A or -1[+]-1[ζ]-A-2[+] or-1[+]-1[ζ]-A-1[+]-1[ζ]-A-1[+] or -1[+]-2[ζ]-A-1[+] or -1[+]-S[ζ]-2[+] or-1[+]-2[ζ]-1[+]-A or -1[+]-2[ζ]-1[+]-1[ζ]-A-1[+] or-1[+]-2[ζ]-1[ζ]-A-1[+] or -3[ζ]-2[+] or -3[ζ]-1[+]-A or-3[ζ]-1[+]-1[ζ]-A-1[+] or -1[ζ]-2A-1[+]-A or -1[ζ]-2A-2[+] or-1[ζ]-2A-1[+]-1[ζ]-A-1[+] or -2[+]-A-[+]-A or -2[+]-1[ζ]-1[+]-A or-1[+]-1[ζ]-A-1[+]-A or -1[+]-2A-1[+]-1[ζ]-A-1[+] or -1[ζ]-A-1[ζ]-A-1[+]Wherein: [ϕ] = Leu, Phe, Trp, Ile, Met, Tyr, or Val [+] = Lys or Arg [ζ]= Gln, Asn, Thr, or Ser A = Ala G = Gly S = Ser n = an integer from 1 to20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 1 to4, or 1 to 3 The number preceding the square brackets indicate thenumber of contiguous residues (e.g., 3[ϕ] = [ϕ]-[ϕ]-[ϕ]) Underlinedsequences indicate the most commonly occurring consensus sequences,based on the alignment shown in FIG. 49G.

Second, a script was designed and built in the programming language,Python, to randomly generate and filter sequences respecting allparameters (except for predicted isoelectric point (pI, parameter 7),because the source code to calculate this parameter was not available atthe time of preparing the present example). Structural parameters 2, 3,4 (amphipathic alpha-helix, positively-charged surface, and highlyhydrophobic core) described in Table A1 were respected by entering theconsensus sequences set forth in Formulas 1 to 8 and in Table C4(appropriate alternance of Hydrophobic, Cationic, hydrophilic, Ala andGly amino acids in the sequence) into the code, and biochemicalparameters (1), (5), (6), and (8)-(15) described in Table A1 were allindividually included into the code to generate 10 000 variant peptidesequences. These variant peptide sequences correspond to SEQ ID NOs:243-10 242.

Example D Rationally-Designed Peptides Facilitate Escape ofEndosomally-Trapped Calcein

Calcein endosomal escape assays were performed as generally described inExample 2 and characterized fluorescence microscopy (data not shown) andby flow cytometry (results for FSD5 are shown below). FSD18 displayedsimilar results to FSD5 (data not shown).

TABLE D1 Calcein endosome escape assays Peptide Mean exposure PeptideCounts time conc. (±St. Dev.; Mean Cells Peptide (min) (μM) n = 3)Factor HeLa No peptide 0 0  4.94 ± 0.39 1.0 FSD5 1 10 76.31 ± 5.18 15.47.5 56.41 ± 5.33 11.3 5 16.27 ± 1.27 3.0 2.5 12.41 ± 0.92 1.5

The result from fluorescence microscopy and flow cytometry experimentsshowed that rationally-designed peptide shuttle agents facilitate theescape of endosomally-trapped calcein in a dose-dependent fashion,similar to the domain-based peptide shuttle agents.

Example E Rationally-Designed Peptides Increase Transduction Efficiencyin Different Cell Types

Protein transduction assays in different cell types were conducted asgenerally described in Example 3.1a (adherent cells) or Example 3.1b(suspension cells), using rationally-designed peptides at the indicatedconcentrations, 10 μM GFP-NLS as cargo, and at the indicated times,before being characterized by flow cytometry (Example 3.3) andfluorescence microscopy (Example 3.2). The results from the flowcytometry are shown in the tables below. Successful delivery of GFP-NLSto the nucleus of cells was verified by fluorescence microscopy (datanot shown).

TABLE E1 GFP-NLS transduction in HeLa cells Conc. of Incu- Mean % cellsCell viability Peptide GFP- bation with GFP (%) conc. NLS time signal(±St. (±St. Dev.; Cells Peptide (μM) (μM) (min) Dev.; n = 3) n = 3) HeLaNo 0 10 2 0.38 ± 0.05 100 peptide FSD5 10 1 70.5 ± 6.44   76 ± 7.45 8 168.5 ± 5.27   85 ± 6.27 5 2 63.1 ± 4.19 35.5 ± 4.82 FSD9 10 1 73.5 ±5.51 79.5 ± 6.33 8 1 70.2 ± 6.83 82.3 ± 7.16 5 2 58.4 ± 4.93 45.6 ± 3.64FSD10 10 1 73.1 ± 5.24 79.75 ± 6.37  8 1 55.9 ± 5.22 83.42 ± 6.38  5 245.8 ± 4.16 55.61 ± 4.28 

TABLE E2 GFR-NLS transduction in HCC-78 cells (human non-small cell lungcarcinoma) Conc. of Incu- Mean % cells Cell Peptide GFP- bation with GFPsignal viability conc. NLS time (±St. Dev.; (%) Cells Peptide (μM) (μM)(min) n = 3) n = 3) HCC-78 No 0 10 2 0.21 ± 0.03 100 peptide FSD5 10 141.9 ± 3.61 15.9 ± 0.83 8 1 69.3 ± 5.27 87.7 ± 6.52 5 2 34.1 ± 3.57 75.3± 6.18 FSD10 10 1 45.0 ± 4.23 63.1 ± 5.27 8 1 15.7 ± 2.67 76.1 ± 6.19 52 22.8 ± 3.06 83.1 ± 5.99 FSD12 10 1 35.9 ± 3.18 46.5 ± 4.18 8 1 39.7 ±4.08 66.3 ± 6.03 5 2 21.4 ± 2.53 75.1 ± 6.31

TABLE E3 GFP-NLS transduction in NCI-H196 cells (human small cell lungcancer) Peptide Conc. of Incubation Mean % cells with Cell viabilityconc. GFP-NLS time GFP signal (%) Cells Peptide (μM) (μM) (min) (±St.Dev.; n = 3) n = 3) NCI-H196 No 0 10 2 0.1 ± 0.02 100 peptide FSD5 10 1 16 ± 1.27 47.19 ± 3.54 8 1 9.1 ± 0.99 69.94 ± 6.38 5 2 7.3 ± 0.82 77.19± 6.17 FSD10 10 1 8.3 ± 0.76 85.44 ± 7.66 8 1 7.4 ± 0.83 80.97 ± 8.02 52 6.4 ± 0.71 83.22 ± 7.51 FSD12 10 1 6.3 ± 0.68 72.52 ± 6.29 8 1 4.5 ±0.38 71.86 ± 6.44 5 2 5.1 ± 0.42 76.51 ± 6.37

TABLE E4 GFP-NLS transduction in THP-1 cells Conc. of Incu- Mean % cellsCell viability Peptide GFP- bation with GFP (%) conc. NLS time signal(±St. (±St. Dev.; Cells Peptide (μM) (μM) (min) Dev.; n = 3) n = 3)THP-1 No 0 10 1.5 0.27 ± 0.01 100 peptide FSD10 1 1 42.6 ± 4.29 93.5 ±5.64 1 1.5 59.4 ± 3.61 78.4 ± 6.15 2 0.5 60.5 ± 5.27 96.3 ± 2.16 2 171.9 ± 5.63 85.6 ± 5.22 FSD18 1 1 43.6 ± 3.55 95.3 ± 3.11 1 1.5 41.7 ±2.82 86.5 ± 6.27 2 0.5 53.4 ± 4.29 97.9 ± 1.73 2 1 78.3 ± 5.48 98.6 ±0.37 FSD19 2 0.5 55.4 ± 4.63 68.7 ± 4.29 5 0.25 61.5 ± 6.07 60.5 ± 5.71FSD21 2 0.5 47.1 ± 3.83 75.6 ± 6.38 5 0.25 57.3 ± 4.52 62.5 ± 5.16 FSD252 0.5 51.9 ± 6.39 79.7 ± 6.52 5 0.25 51.5 ± 4.17 66.9 ± 5.17

TABLE E5 GFP-NLS transduction in various suspension cells Cell viabilityPeptide Conc. of Incubation Mean % cells with (%) conc. GFP-NLS time GFPsignal (±St. Dev.; Peptide Cells (μM) (μM) (min) (±St. Dev.; n = 3) n =3) No [All] 0 10 90  0.36 ± 0.03* 100* peptide FSD18 DOHH2 1 90  2.4 ±0.42 62.5 ± 6.17 2 30 26.8 ± 2.19 79.8 ± 6.18 10 27.2 ± 2.46 25.0 ± 2.66HT2 1 90 12.3 ± 0.96 88.3 ± 7.91 2 30 31.0 ± 2.55 80.7 ± 7.10 10 82.5 ±4.07 63.9 ± 5.35 Jurkat 1 90 10.1 ± 1.11 98.6 ± 0.39 2 30 11.0 ± 1.2997.4 ± 1.09 10  9.9 ± 1.06 96.6 ± 2.46 KMS-12BM 1 90 13.6 ± 2.17 97.6 ±1.05 2 30 26.2 ± 3.93 95.1 ± 3.56 10 21.0 ± 1.76 92.7 ± 4.11 REC-1 1 901.80 ± 0.88 96.2 ± 2.53 2 30 10.9 ± 1.34 99.0 ± 0.39 10 25.0 ± 1.89 25.0± 3.17 NK 1 90 2.80 ± 0.33 99.1 ± 0.08 2 30 6.41 ± 1.12 98.3 ± 1.00 5 1565.7 ± 5.27 94.9 ± 1.63 *Quantification of the negative control (“nopeptide”) was similar for all cell lines tested. Thus, the data (*)represents an average from the “no peptide” controls for at cell linestested.

Example F Rationally-Designed Peptide Shuttle Agents Enable Transductionof Antibodies F.1 Transduction of Fluorescently Labeled Antibodies byFSD5 in HeLa Cells

Protein transduction assays were conducted as generally described inExample 3.1, using the peptide FSD5 and an antibody as cargo after 1 minincubation time, before being characterized by fluorescence microscopy(Example 3.2). FIG. 50 shows the results of the cytoplasmic transductionof Goat Anti-Mouse IgG H&L (Alexa Fluor® 488) and Goat Anti-Rabbit IgGH&L (Alexa Fluor® 594) antibodies delivered in HeLa cells by the peptideFSD5 (8 μM) for 1 min and visualized by fluorescence microscopy at 20×magnification for the Alexa Fluor 594 Ab (FIG. 50A); and at 10× and 20×magnification for the Alexa Fluor 488 Ab (FIGS. 50B and 50C,respectively). The bright field and fluorescence images of living cellsare shown in upper and lower panels, respectively.

The following experiments show that other FSD peptides can also deliverfunctional antibodies: an anti-NUP98 antibody which labels the nuclearmembrane, and two anti-Active Caspase3 antibodies that bind andinactivate the pro-apoptotic Caspase 3 protein. The delivery, microscopyand cell immune-labelling protocols are described in Example 3.

F.2 Transduction of Anti-NUP98 Antibody by FSD19 in HeLa Cells

Anti-NUP98 antibody (10 μg) was co-incubated with 7.5 μM of FSD19 andexposed to HeLa cells for 4 hours. Cells are washed, fixed withparaformaldehyde 4%, permeabilized with 0.1% Triton™ and labeled with afluorescently labeled (Alexa™ Fluor 488) goat anti-rat antibody.Antibody bound to the perinuclear membrane and cell nuclei werevisualized by fluorescence microscopy at 20× (upper panels) and 40×(lower panels). As shown in FIG. 500, green fluorescent signal emanatedfrom the nuclear membrane (left panels) and overlapped with Hoechststaining (right panels), demonstrating that the anti-NUP98 antibodyretained its functionality inside the cell following its transduction.

F.3 Transduction of Two Functional Anti-Active Caspase 3 Antibodies byFSD23 in THP-1 and Jurkat Cells: Quantification by ELISA Cleaved PARPAssay

A monoclonal (mAb) and a polyclonal (pAb) anti-Active Caspase 3antibodies (2 μg) were independently co-incubated with THP-1 and Jurkatcells for 5 min in the presence of FSD23 at 7.5 μM. The anti-apoptoticeffect of each antibody was assessed via the level of Caspase3-activated apoptosis with an ELISA cleaved PARP assay and quantified byspectrometry as described below.

The day of the experiment, cells in exponential growth phase wereharvested, centrifugated (400 g for 3 min) and resuspended in serum-freeRPMI in a 96-well plate (500,000 cells in 150 μL per well). Cells werecentrifugated and incubated for 5 min with a mix composed by the peptideto be tested (7.5 μM) and 2 μg of the antibody to be transduced. Cellswere centrifuged and resuspended in RPMI with serum in a 24-well platefor 1 h at 37° C. Actinomycin D (2 μg/mL), a cytotoxic inducer ofapoptosis, was incubated with the cells for 4 h. Cells were washed withcold PBS and tested using the PARP (Cleaved) [214/215] Human ELISA Kit(ThermoFisher) according to the manufacturer's instructions followed byspectroscopy analysis. Results are shown in Table F1.

TABLE F1 Cleaved PARP ELISA assay after transduction of anti-TNF oranti-Active Caspase 3 antibody by FSD23 in THP-1 and Jurkat cellsOptical Density (O.D.) PARP Cell type Antibody Actinomycin D cleavageassay THP-1 anti-TNF − 0.334 (control) + 1.162 anti-Active − 0.207Caspase 3 mAb + 0.856 anti-Active − 0.192 Caspase 3 pAb + 0.653 Jurkatanti-TNF − 0.281 (control) + 0.486 anti-Active − 0.174 Caspase 3 mAb +0.301 anti-Active − 0.149 Caspase 3 pAb + 0.333

Results in THP-1 and in Jurkat cells show that FSD23 successfullytransduced functional anti-Active Caspase 3 antibodies. Anti-TNFantibody was used as a non-specific negative control and actinomycin Das a cytotoxic inducer of apoptosis. In the absence of actinomycin D(“−”), the delivery of each anti-Active Caspase 3 mAb and pAb resultedin the reduction of the basal level of apoptosis compared to the“anti-TNF” control, in which the delivery of the anti-TNF antibody hadno discernable impact on cell viability. In presence of actinomycin D(“+”), the resulting apoptosis was reduced after the delivery of bothanti-Active Caspase 3 antibodies with FSD23 compared to the “anti-TNF”control.

Example G Rationally-Designed Peptide Shuttle Agents Enable Transductionof CRISPR-Based Genome Editing Complexes

We tested the ability of rationally-designed peptide shuttle agents todeliver functional CRISPR-based genome editing complexes to the nucleusof eukaryotic cells using standard DNA cleavage assays. These assayswere used to measure CRISPR/Cas9 and CRISPR/Cpf1-mediated cleavage ofcellular genomic DNA sequences HPRT (HypoxanthinePhosphoribosyltransferase 1) and DNMT1 (DNA(Cytosine-5-)-Methyltransferase 1), respectively. Homologous-directedrecombination (HDR) of short (72 bp) and long (1631 bp) DNA templateswere performed at the HPRT genomic cut site, and measured afterintracellular delivery of the genome editing complexes with differentshuttle agents.

G.1 CRISPR/Cas9-NLS Complex Transduction by Rationally-Designed PeptideShuttle Agents, Cleavage of Genomic Target Sequence, andHomologous-Directed Recombination in Different Cell Lines G.1.1Transduction of Functional CRISPR/Cas9-NLS Complexes

Cas9-NLS recombinant protein was prepared as described in Example 13.1.A mix composed of a Cas9-NLS recombinant protein and crRNA/tracrRNA (seebelow) targeting a nucleotide sequence of the HPTR genes wereco-incubated with different concentrations of FSD5, FSD8, FSD10 or FSD18and incubated with HeLa, HCC-78, NIC-H196 or REC-1 cells for 2 min inPBS, or 48 h in medium with serum, using the transduction protocols asgenerally described in Example 3.1a. Cells were then washed with PBS andharvested to proceed with the T7E1 protocol assay as described inExample 13.4.

The sequences of the crRNA and tracrRNAs constructed and their targetswere:

Feldan tracrRNA [SEQ ID NO: 77]:5′-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3′ HPRT crRNA [SEQ ID NO: 103]:5′-AAUUAUGGGGAUUACUAGGAGUUUUAGAGCUAUGCU-3′

FIGS. 51A-51F show the results of the cleavage of the targeted genomicHPRT DNA sequence with the CRISPR/Cas9 (2.5 μM) and the crRNA/tracrRNA(2 μM) in the absence (“− ctrl”) or presence of the shuttle agents FSD5,FSD8, FSD10 or FSD18 used at different concentrations, exposure times,and in different types of cells: HeLa (FIGS. 51A and 51B); NK (FIG.51C); NIC-196H (FIG. 51D); HCC-78 (FIG. 51E) and REC-1 cells (FIG. 51F),after separation by agarose gel electrophoresis. In some cases, gellanes were loaded in duplicate. Thin dashed arrows indicate the bandscorresponding to the target gene, and thicker solid arrows indicate thebands corresponding to the cleavage products of this target gene, whichindicate the successful transduction of functional CRISPR/Cas9-NLSgenome editing complexes. We used a Bio-Rad ImageLab™ software (Version5.2.1, Bio-Rad,http://www.bio-rad.comlen-ca/product/image-lab-software?tab=Download) toquantify the relative signal intensities of each of the different bandsdirectly on gels. The sum of all the bands in a given lane correspondsto 100% of the signal, and the numerical value in italics at the bottomof each lane is the sum of the relative signals (%) of only the twocleavage product bands (thicker solid arrows). No cleave product bandswere found in the negative controls (“− ctrl”, i.e., to cells that wereexposed to CRISPR/Cas9-NLS complex in the absence of shuttle agent).These results indicate the successful delivery of the CRISPRgenome-editing complexes to the nucleus, resulting in cleave of thetarget gene.

G.1.2 Transduction of CRISPR/Cas9-NLS Complexes with Short Linear DNATemplate, Resulting in Homologous-Directed Recombination

A mix was prepared containing: a Cas9-NLS recombinant protein (2.5 μM)(see Example 13.1); the crRNA/tracrRNA (2 μM) targeting a nucleotidesequence of the HPTR genes (see above); the peptide shuttle agent FSD5(15 μM); and either 0 ng or 500 ng of a short linear template DNA (72bp; see below).

Short DNA template [SEQ ID NO: 154]:5′-TGAAATGGAGAGCTAAATTATGGGGATTACAAGCTTGATAGCGAAGGGGCAGCAATGAGTTGACACTACAGA-3′

This mixture was exposed to HeLa cells for 48 h in culture mediacontaining serum. Cells were then washed and subjected to the T7E1 assayas described in Example 13.4.

FIG. 51G shows the cleavage of the targeted HPRT genomic sequence by theCRISPR/Cas9 complex transduced by FSD5 (15 μM), in the absence (“Notemplate”) or presence (+500 ng) of the short DNA template. Thin dashedarrows indicate the bands corresponding to the target gene, and thickersolid arrows indicate the bands corresponding to the cleavage productsof this target gene, which indicate the successful transduction of fullyfunctional genome editing complexes. The numerical value in italics atthe bottom of each lane is the sum of the relative signals (%) of onlythe two cleavage product bands (thicker solid arrows). These resultsshow that FSD5 can transduce a functional CRISPR/Cas9 complex in thepresence or absence of template DNA.

To verify whether homologous-directed recombination occurred, we usedthe genomic DNA extracted from FSD5/CRISPR/short DNA template-treatedcells to amplify the short DNA template sequence with specificallydesigned oligonucleotide primers targeting this sequence. Theamplification of the short DNA template sequence confirmed the insertionof this template in the genome after the cutting of the HPRT gene by theCRISPR/Cas9-NLS genome editing complex. The PCR products were resolvedby agarose gel electrophoresis and the results are shown in FIG. 51H. Noamplification was detected in the “no template” sample, in which thegenomic DNA was cut but no DNA template was provided (FIG. 51H). Incontrast, an amplicon of appropriate size (FIG. 51H, thick solid line)was detected for the “+500 ng” sample, in which the genomic DNA was cutand a DNA template was provided. Detection of the amplicon indicatessuccessful insertion of the short DNA template sequence into the genome.These results show that FSD5 can transduce CRISPR/Cas9 complex in thepresence of a short DNA template, resulting in homologous-directedrecombination.

G.1.3 Transduction of CRISPR/Cas9-NLS Complexes with Long Linear DNATemplate, Resulting in Homologous-Directed Recombination

A mix was prepared containing: a Cas9-NLS recombinant protein (2.5 μM)(see Example 13.1); the crRNA/tracrRNA (2 μM) targeting a nucleotidesequence of the HPTR genes (see above); the peptide shuttle agent FSD5(15 μM); and either 0 ng or 500 ng of a long linear template DNAencoding GFP (1631 bp; see below).

GFP coding DNA template [SEQ ID NO: 156]:5′AAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCCGCTAGCGCTACCGGTCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTCCGGACTCAGATCTCGAGCTCAAGCTTCGAATTCTGCAGTCGACGGTACCGCGGGCCCGGGATCCACCGGATCTAGATAACTGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTAA-3′

This mixture was exposed to HeLa cells for 48 h in culture mediacontaining serum. Cells were then washed with PBS and harvested toproceed with the T7E1 protocol assay as described in Example 13.4.

FIG. 51I shows the cleavage of the targeted genomic HPRT genomicsequence by the CRISPR/Cas9 complex transduced by FSD5 (15 μM), in theabsence (“No template”) or presence (“+500 ng”) of the long DNAtemplate. The cleavage products are indicated with thick solid arrows.These results show that FSD5 can transduce a functional CRISPR/Cas9complex in the presence or absence of a long template DNA.

To verify whether homologous-directed recombination occurred, we usedthe genomic DNA extracted from FSD5/CRISPR/long DNA template-treatedcells to amplify the long DNA template sequence with specificallydesigned oligonucleotide primers flanking this sequence. Theamplification of the long DNA template sequence confirmed the insertionof this template in the genome after the cutting of the HPRT gene by theCRISPR/Cas9-NLS genome editing complex. The PCR products were resolvedby agarose gel electrophoresis and the results are shown in FIG. 51J. Inthe “No template” sample, a single band corresponding to the ampliconlacking the long DNA template insertion was detected. In contrast,additional larger bands (indicated with an arrow) were detected for the“+500 ng” (faint) and “+1000 ng” (darker) samples, indicating someinsertion of the long DNA template into the genomic DNA had occurred.These results show that FSD5 can transduce CRISPR/Cas9 complex in thepresence of a long DNA template, resulting in homologous-directedrecombination.

G.2 CRISPR/Cpf1-NLS Complex Transduction by Rationally-Designed ShuttleAgents, Cleavage of Genomic Target Sequence in HeLa and NK Cells

A mix composed of a Cpf1-NLS recombinant protein (2.5 μM) and crRNA (2μM; see below) targeting a nucleotide sequence of the DNMT1 gene wasco-incubated with different concentrations of FSD18 and incubated withHeLa or NK cells for 2 min in HeLa cells, or 90 sec in NK cells in PBSor in medium without serum using transduction protocols as described inExample 3.1a.

The sequence of the Cpf1-NLS recombinant protein produced was:

[SEQ ID NO: 155] MTQFEGFTNLYQVSKTLRFELIPQGKTLKHIQEQGFIEEDKARNDHYKELKPIIDRIYKTYADQCLQLVQLDWENLSAAIDSYRKEKTEETRNALIEEQATYRNAIHDYFIGRTDNLTDAINKRHAEIYKGLFKAELFNGKVLKQLGTVTTTEHENALLRSFDKFTTYFSGFYENRKNVFSAEDISTAIPHRIVQDNFPKFKENCHIFTRLITAVPSLREHFENVKKAIGIFVSTSIEEVFSFPFYNQLLTQTQIDLYNQLLGGISREAGTEKIKGLNEVLNLAIQKNDETAHIIASLPHRFIPLFKQILSDRNTLSFILEEFKSDEEVIQSFCKYKTLLRNENVLETAEALFNELNSIDLTHIFISHKKLETISSALCDHWDTLRNALYERRISELTGKITKSAKEKVQRSLKHEDINLQEIISAAGKELSEAFKQKTSEILSHAHAALDQPLPTTLKKQEEKEILKSQLDSLLGLYHLLDWFAVDESNEVDPEFSARLTGIKLEMEPSLSFYNKARNYATKKPYSVEKFKLNFQMPTLASGWDVNKEKNNGAILFVKNGLYYLGIMPKQKGRYKALSFEPTEKTSEGFDKMYYDYFPDAAKMIPKCSTQLKAVTAHFQTHTTPILLSNNFIEPLEITKEIYDLNNPEKEPKKFQTAYAKKTGDQKGYREALCKWIDFTRDFLSKYTKTTSIDLSSLRPSSQYKDLGEYYAELNPLLYHISFQRIAEKEIMDAVETGKLYLFQIYNKDFAKGHHGKPNLHTLYWTGLFSPENLAKTSIKLNGQAELFYRPKSRMKRMAHRLGEKMLNKKLKDQKTPIPDTLYQELYDYVNHRLSHDLSDEARALLPNVITKEVSHEIIKDRRFTSDKFFFHVPITLNYQAANSPSKFNQRVNAYLKEHPETPIIGIDRGERNLIYITVIDSTGKILEQRSLNTIQQFDYQKKLDNREKERVAARQAWSVVGTIKDLKQGYLSQVIHEIVDLMIHYQAVVVLENLNFGFKSKRTGIAEKAVYQQFEKMLIDKLNCLVLKDYPAEKVGGVLNPYQLTDQFTSFAKMGTQSGFLFYVPAPYTSKIDPLTGFVDPFVWKTIKNHESRKHFLEGFDFLHYDVKTGDFILHFKMNRNLSFQRGLPGFMPAWDIVFEKNETQFDAKGTPFIAGKRIVPVIENHRFTGRYRDLYPANELIALLEEKGIVFRDGSNILPKLLENDDSHAIDTMVALIRSVLQMRNSNAATGEDYINSPVRDLNGVCFDSRFQNPEWPMDADANGAYHIALKGQLLLNHLKESKDLKLQNGISNQDWLAYIQELRNGGRSSDDEATADSQHAAPPKKKRKV GGSGGGSGGGSGGGRHHH HHH(MW = 155.7 kDa; pI = 8.34) NLS sequence is underlinedSerine/glycine rich linkers are in bold

The sequences of the crRNA used was as follows:

DNMT1 crRNA [SEQ ID NO: 157]:5′-AAUUUCUACUGUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC-3′

After 2 min (HeLa) or 90 sec (NK), cells were washed with PBS andharvested to proceed with the T7E1 protocol assay as described inExample 13.4. The PCR-amplified DNMT1 DNA sequence and the PCR-amplifiedcleavage product of this sequence were resolved on agarose gels and theresults are shown in FIGS. 51K (HeLa cells) and 51L (NK cells). Thenegative control (“− ctrl”) corresponds to cells that were exposed toCRISPR/Cpf1-NLS complex in the absence of the shuttle agent Thin dashedarrows indicate the bands corresponding to the target gene, and thicksolid arrows indicate the bands corresponding to the cleavage productsof this target gene, which indicate the successful transduction of fullyfunctional CRISPR/Cpf1-NLS genome editing complexes. The numerical valuein italics at the bottom of each lane is the sum of the relative signals(%) of only the two cleavage product bands (thick solid arrows). Theseresults show that FSD18 can transduce a functional CRISPR/Cpf1-NLScomplex into the nucleus of these cells to effect cleavage of the targetgene.

The CRISPRMAX™ technology is a commercially availablelipofectamine-based transfection reagent optimized for CRISPR-Cas9protein delivery. However, an equivalent reagent does not presentlyexist for the transduction of CRISPR-Cpf1. Interestingly, when we usedthe CRISPRMAX™ reagent, it was unable to deliver the CRISPR/Cpf1-NLScomplex in adherent and suspension cells. In contrast, FSD18 enabled arobust cleavage of the DNMT1 target in HeLa cells, and a lower butobservable cleavage in NK cells.

These results show that the shuttle agent FSD18 successfully delivered afunctional CRISPR/Cpf1-NLS complex to the nucleus of HeLa and NK cells,and that this delivery resulted in a CRISPR/Cpf1-NLS-mediated cleavageof genomic DNA.

Examples G.3-G.10 Rationally-Designed Peptide Shuttle Agents EnableSingle or Multiple Gene Targeting, and/or Co-Delivery of DifferentCRISPR-Based Genome Editing Complexes

These examples support the ability of rationally-designed peptideshuttle agents to enable the delivery and edition of multiple genetargets simultaneously. Functional CRISPR-based genome editing complexeswere delivered to the nucleus of eukaryotic cells, and successful genomeediting was evaluated using standard DNA cleavage assays. These assayswere used to measure CRISPR/Cas9-mediated cleavage of cellular genomicDNA sequences HPRT (Hypoxanthine Phosphoribosyltransferase 1) and B2M(02 microglobulin HLA subunit), and to measure CRISPR/Cpf1-mediatedcleavage of cellular genomic DNA sequences NKG2A (Inhibitory NK cellreceptor 2A), GSK3 (Glycogen Synthase Kinase 3), CBLB (E3Ubiquitin-protein Ligase), DNMT1 (DNA (Cytosine-5-)-Methyltransferase 1)and B2M (β2 microglobulin HLA subunit). We also performed more complexgenome editing approaches with the delivery of multiple CRISPR systemstargeting one or two genes in the same cells. CRISPR/Cas9 andCRISPR/Cpf1 complexes were delivered together in HeLa cells to edit theHPRT and DNMT1 genes, respectively, or to edit the B2M gene in twodifferent loci of exon 2. Finally, we co-delivered two CRISPR/Cpf1complexes, each carrying a specific crRNA, to edit two exons in the B2Mgene in NK cells.

G.3 Different Rationally-Designed Peptide Shuttle Agents DeliverCRISPR/Cas9-NLS and CRISPR/Cpf1 Complexes for B2M Gene Editing in HeLa,THP-1 and NK Cells

Cas9-NLS recombinant protein was prepared as described in Example 13.1.Cpf1-NLS recombinant protein was prepared as described in Example G.2. Amix composed of a Cas9-NLS recombinant protein with its respectivecrRNA/tracrRNA, or a Cpf1-NLS recombinant protein with its respectivesingle guide crRNA(s) (see below) targeting a nucleotide sequence of theB2M gene, was co-incubated with different concentrations of the peptidesFSD10, FSD18, FSD19, FSD21, FSD22, or FSD23 and incubated with HeLa,THP-1 or NK cells for 90 sec in PBS, or for 1 h in medium without serum,or for 48 h in medium with serum, using the transduction protocols asgenerally described in Example 3.1a. Cells were then washed with PBS andharvested to proceed with the T7E1 protocol assay as described inExample 13.4.

The sequences of the crRNAs and tracrRNAs constructed and their targetswere:

Feldan tracrRNA [SEQ ID NO: 77]:5′-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3′ Cas9-f1anked B2M-crRNA [SEQ ID NO: 160]:5′-GAGTAGCGCGAGCACAGCTAGUUUUAGAGCUAUGCUGUUUUG-3′Cpf1-flanked B2M crRNA-1 [SEQ ID NO: 161]:5′-AAUUUCUACUGUUGUAGAUAUCCAUCCGACAUUGAAGUU-3′Cpf1-flanked B2M crRNA-2 [SEQ ID NO: 162]:5′-AAUUUCUACUCUUGUAGAUCCGAUAUUCCUCAGGUACUCCA-3′

FIGS. 52A-52D show the results of the cleavage of the targeted genomicB2M DNA sequence after the delivery of CRISPR/Cpf1 (1.33 μM) withcrRNA-1 or crRNA-2 (2 μM) in the absence (“− ctrl”) or in the presenceof the peptides FSD10, FSD18, FSD19, FSD21 or FSD23 used at differentconcentrations, exposure times, and in different types of cells: THP-1(FIG. 52A), and NK (FIGS. 52B, 52C, 52D), after separation by agarosegel electrophoresis. FIG. 52D shows cleavage products of the genomic B2Mexon 2 DNA sequence after the delivery of a CRISPR/Cpf1 complex carryinga specific single guide RNA (crRNA-1 or crRNA-2) in presence of FSD18 orFSD21, respectively. FIG. 52E shows the cleavage product of the genomicB2M exon 2 DNA sequence with CRISPR/Cas9 (2.5 μM) and crRNA (2 μM) inthe absence (“− ctrl”) or in the presence of the peptide FSD22 used at10 μM for 1 h in HeLa cells, after separation by agarose gelelectrophoresis. Gel lanes were loaded in duplicate. Thin dashed arrowsindicate the bands corresponding to the target gene, and thick solidarrows indicate the bands corresponding to the cleavage products of thistarget gene, which indicate the successful transduction of fullyfunctional CRISPR genome editing complexes. We used a Bio-Rad ImageLab™software (Version 5.2.1, Bio-Rad,http://www.bio-rad.com/en-ca/product/image-lab-software?tab-Download) toquantify the relative signal intensities of each of the different bandsdirectly on the gels. The sum of all the bands in a given lanecorresponds to 100% of the signal, and the numerical value in italics atthe bottom of each lane is the sum of the relative signals (%) of onlythe two cleavage product bands (thick solid arrows). No cleavage productbands were found in the negative controls (“− ctrl”, i.e., to cells thatwere exposed to CRISPR system in the absence of FSD peptide). Theseresults indicate the successful delivery of the CRISPR genome-editingcomplexes to the nucleus, resulting in cleavage of the target gene.

G.4 Different Rationally-Designed Peptide Shuttle Agents DeliverCRISPR/Cpf1 Systems for GSK3, CBLB and DNMT1 Gene Editing in NK, THP-1and Primary Myoblasts Cells.

Cpf1-NLS recombinant protein was prepared as described in Example G.2. Amix composed of a Cpf1-NLS recombinant protein with a single guide crRNA(see below) targeting a nucleotide sequence of the GSK3, CBLB or DNMT1genes was co-incubated with different concentrations of FSD10, FSD18,FSD19 or FSD23 and incubated with NK cells for 48 h in medium withserum, and in THP-1 or in primary myoblasts cells for 90 sec in PBS,using the transduction protocols as generally described in Example 3.1a.Cells were then washed with PBS and harvested to proceed with the T7E1protocol assay as described in Example 13.4.

The sequences of the crRNA constructed and their targets were:

GSK3 crRNA [SEQ ID NO: 163]:5′-AAUUUCUACUCUUGUAGAUCUUUCUUCCUUUAGGAGACA-3′CBLB crRNA [SEQ ID NO: 164]:5′-AAUUUCUACUCUUGUAGAUAAGAACUAAAAUUCCAGAUG-3′DNMT1 crRNA [SEQ ID NO: 157]:5′-AAUUUCUACUGUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC-3′

FIGS. 52F-52I show the results of the cleavage of the targeted genomicGSK3, CBLB and DNMT1 DNA sequences with the CRISPR/Cpf1 (1.33 μM) andcrRNA (2 μM) in absence (“− ctrl”) or presence of the shuttle agentsFSD10, FSD18, FSD19 or FSD23 used at different concentrations, exposuretimes, and in different types of cells: NK (FIGS. 52F and 52G), THP-1(FIG. 52H) and primary myoblasts (FIG. 52I) after separation by agarosegel electrophoresis. Gel lanes were loaded in duplicate. Thin dashedarrows indicate the bands corresponding to the target gene, and thicksolid arrows indicate the bands corresponding to the cleavage productsof this target gene, which indicate the successful transduction of fullyfunctional CRISPR genome editing complexes.

G.5 Different Rationally-Designed Peptide Shuttle Agents DeliverCRISPR/Cpf1 Systems for NKG2A Gene Editing in NK Cells.

Cpf1-NLS recombinant protein was prepared as described in Example G.2. Amix composed of a Cpf1-NLS recombinant protein with a single guide crRNA(see below) targeting a nucleotide sequence of the NKG2A gene wasco-incubated with different concentrations of FSD10, FSD21, FSD22 orFSD23 and incubated with NK and NK-92 cells for 90 sec in PBS, using thetransduction protocols as generally described in Example 3.1a. Cellswere then washed with PBS and harvested to proceed with the T7E1protocol assay as described in Example 13.4.

The sequences of the crRNA constructed and their targets were:

NKG2A crRNA [SEQ ID NO: 165]:5′-AAUUUCUACUCUUGUAGAUGGGGCAGAUUCAGGUCUGAG-3′

FIGS. 52J-52N show the results of the cleavage of the targeted genomicNKG2A DNA sequence with the CRISPR/Cpf1 (1.33 μM) and crRNA (2 μM) inabsence (“− ctrl”) or presence of the shuttle agents FSD10, FSD21, FSD22or FSD23 used at different concentrations, exposure times, and in NK andNK-92 cells after separation by agarose gel electrophoresis. Gel laneswere loaded in duplicate. Thin dashed arrows indicate the bandscorresponding to the target gene, and thick solid arrows indicate thebands corresponding to the cleavage products of this target gene, whichindicate the successful transduction of fully functional CRISPR genomeediting complexes.

G.6 Different Rationally-Designed Peptide Shuttle Agents Co-DeliverCRISPR/Cas9 and CRISPR/Cpf1 Complexes for HPRT, DNMT1 and B2M GeneEditing in HeLa and NK Cells

Cas9-NLS recombinant protein was prepared as described in Example 13.1.Cpf1-NLS recombinant protein was prepared as described in Example G.2. Amix composed of Cas9-NLS recombinant protein with its respectivecrRNA/tracrRNA, or Cpf1-NLS recombinant protein with its respectivesingle guide crRNA(s) (see below) targeting a nucleotide sequence of theDNMT1, HPRT and B2M genes, was co-incubated with differentconcentrations of FSD10, FSD18, FSD21 or FSD23, and incubated with HeLaor NK cells for 90 sec or 2 min in PBS using the transduction protocolsas generally described in Example 3.1a. Cells were then washed with PBSand harvested to proceed with the T7E1 protocol assay as described inExample 13.4.

The sequences of the crRNA and tracrRNAs constructed and their targetswere:

Feldan tracrRNA [SEQ ID NO: 77]:5′-AAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGCU-3′ HPRT crRNA [SEQ ID NO: 103]:5′-AAUUAUGGGGAUUACUAGGAGUUUUAGAGCUAUGCU-3′ DNMT1 crRNA [SEQ ID NO: 157]:5′-AAUUUCUACUGUUGUAGAUCUGAUGGUCCAUGUCUGUUACUC-3′Cas9-f1anked B2M-crRNA [SEQ ID NO: 160]:5′-GAGUAGCGCGAGCACAGCTAGUUUUAGAGCUAUGCUGUUUUG-3′Cpf1-flanked B2M crRNA-1 [SEQ ID NO: 161]:5′-AAUUUCUACUGUUGUAGAUAUCCAUCCGACAUUGAAGUU-3′Cpf1-flanked B2M crRNA-2 [SEQ ID NO: 162]:5′-AAUUUCUACUCUUGUAGAUCCGAUAUUCCUCAGGUACUCCA-3′

FIGS. 53A-53C show the results of the cleavage of the targeted genomicDNMT1, HPRT and B2M DNA sequences with different CRISPR systems in theabsence (“− ctrl”) or in the presence of the shuttle agents FSD10,FSD18, FSD21 or FSD23 used at different concentrations, exposure times,and in HeLa and NK cells after separation by agarose gelelectrophoresis. FIG. 53A shows DNMT1 (left panel) and HPRT (rightpanel) DNA cleavage products from the same genomic DNA extract after theco-delivery of a DNMT1-targeting CRISPR/Cpf1 (1.25 μM) complex and aHPRT-targeting CRISPR/Cas9 (1.25 μM) complex in HeLa cells. FIG. 53Bshows the cleavage products of the B2M exon 2 from the same genomic DNAextract after the co-delivery of a CRISPR/Cpf1 (1.25 μM) and aCRISPR/Cas9 (1.25 μM) in HeLa cells. Each complex targeted a differentlocus in the B2M exon 2 via a specific crRNA flanking Cpf1 (left panel)or a specific crRNA flanking Cas9 (Right panel). FIG. 53C shows theresults of the cleavage of the B2M exon 2 from genomic extracts afterthe co-delivery of CRISPR/Cpf1 (1.33 μM) complexes, each one carrying aspecific single guide crRNA-1 or crRNA-2 (2 μM) in presence of FSD10(upper panel), FSD21 (middle panel) or FSD23 (bottom panel). For eachexperiment, NK cells were exposed to CRISPR/Cpf1 with crRNA-1 orCRISPR/Cpf1 with crRNA-2, or both complexes.

G.7 Different Rationally-Designed Peptide Shuttle Agents DeliverCRISPR/Cpf1 Complexes for B2M Gene Editing in T Cells—Flow CytometryAnalysis

Cpf1-NLS recombinant protein was prepared as described in Example G.2.

Unless otherwise specified, T cells used herein were obtained fromhealthy human blood collected in heparinized tubes. T cells wereisolated using a Ficoll™ technique (Ficoll-Paque™ GE or Lymphoprep™ StemCell Technologies). Briefly, blood was mixed with the Ficoll™ solutionin conical tubes (50 mL) and centrifuged at 2280 rpm for 20 minutes.Mononuclear cells were harvested and transferred in another conical tube(50 mL) before washing with PBS and centrifugation at 1100 rpm for 10minutes. Cells were resuspended in 5 mL of PBS containing 20% FBS. Cellswere counted and then incubated in a culture medium composed by RPMIadvanced (cat 12633012 ThermoFisher), 10% FBS, 1% Penstrep (15140122ThermoFisher), 1% L-glutamine (25030081 ThermoFisher) IL-2 30 U/ml).Next, T cells were enriched with a Human T cell Enrichment Kit (StemCell# cat 19051) by negative selection following the manufacturerinstructions. The enriched T cells were validated using a specificanti-CD3 antibody (Biolegend # cat: 300438). At this step, collectedcells were typically around 99% T cells. T cells were activated byadding IL-2 at 30 U/mL and the anti-CD28 antibody (ThermoFisher # cat16-0289-85) in complete medium for 5 days prior to experimentation. Theactivation of T cell expansion was then double-checked with bothanti-CD25 and anti-CD137 antibodies.

A mix composed of a Cpf1-NLS recombinant protein with respective singleguide crRNA(s) targeting a nucleotide sequence of the B2M gene wasco-incubated with different concentrations of FSD21 or FSD18 peptideshuttle agents and incubated with T cells for 90 seconds in PBS usingthe transduction protocols as generally described in Example 3.1a. Eachof the B2M crRNAs were designed to mediate CRISPR/Cpf1-based cleavage ofthe B2M gene, the phenotypic effects of which can be seen by thedisruption of cell surface HLA, which is detectable by flow cytometryusing a fluorescent APC Mouse Anti-Human HLA-ABC antibody.

The cells were then resuspended in 100 μL PBS containing 1% FBS and 4 μLof APC Mouse Anti-Human HLA-ABC antibody before an incubation period of20 minutes, in the dark, at ambient temperature. Then, 1 mL of PBScontaining 1% FBS was added to the suspension followed by a 1200 rpmcentrifugation of 5 minutes. Finally, the pellet was resuspended in 100to 200 μL of PBS containing 1% FBS before flow cytometry analysis.

Flow cytometry results based on cell size and granularity usingrespectively the Forward Scatter (FSC) and the Side Scatter (SSC)parameters showed that viability of the transduced T cells was notsubstantially affected by the co-delivery of different testedconcentrations of FSD21 or FSD18 peptide shuttle agents with CRISPR/Cpf1systems (data not shown).

FIGS. 54A-54D and 55A-55D show delivery of CRISPR/Cpf1 genome editingcomplexes via the shuttle peptides FSD21 and FSD18, respectively. Asseen in FIGS. 54A and 55A, “untreated” negative control cells, whichwere not exposed to CRISPR/Cpf1 or shuttle peptide, exhibited nosignificant genome editing (lack of HLA-negative cells). FIGS. 54B-54Dshow that FSD21 concentrations of 8, 10 and 12 μM resulted in 9.87%,8.68%, and 12.2% of HLA-negative cells, indicating successful nucleardelivery of functional CRISPR/Cpf1 genome editing complexes andsubsequent genome editing. FIGS. 55B-55D show that FSD18 concentrationsof 8, 10 and 12 μM resulted in 8.0%, 9.43%, and 7.9% of HLA-negativecells, indicating successful nuclear delivery of functional CRISPR/Cpf1genome editing complexes and subsequent genome editing.

G.8 Transduction of CRISPR/Cpf1 Complexes Containing Multiple GuidecrRNA Targeting B2M in THP-1 Cell Lines Using a SingleRationally-Designed Peptide Shuttle Agent

Cpf1-NLS recombinant protein was prepared as described in Example G.2. Amix composed of a Cpf1-NLS recombinant protein with a single guide crRNA(see below) targeting one of three chosen nucleotide sequences of theB2M gene was co-incubated with (3 μM) of FSD18 and incubated with THP-1cells for 90 seconds in PBS, using the transduction protocols asgenerally described in Example 3.1a. The same experiments were performedusing a mix composed of a Cpf1-NLS recombinant protein with three guidecrRNA (see below), each targeting three different nucleotide sequencesof the B2M gene. Flow cytometry experiments were performed as describedin Example G.7. Also, to proceed with the T7E1 protocol assay asdescribed in Example 13.4, cells were washed with PBS and harvested,

The sequences of the crRNA constructed and their targets were:

B2M crRNA-E [SEQ ID NO: 166]:5′-AAUUUCUACUCUUGUAGAUAUCCAUCCGACAUUGAAGUU-3′B2M crRNA-J [SEQ ID NO: 167]:5′-AAUUUCUACUCUUGUAGAUCCGAUAUUCCUCAGGUACUCCA-3′B2M crRNA-G [SEQ ID NO: 168]:5′-AAUUUCUACUCUUGUAGAUUUAGAGUCUCGUGAUGUUUAAG-3′

Flow cytometry results based on cell size and granularity usingrespectively the Forward Scatter (FSC) and the Side Scatter (SSC)parameters show that the viability of the transduced THP-1 cells was notsubstantially affected by the presence of CRISPR/Cpf1 systems comprisingthe guide crRNAs (RNA-E, RNA-G, RNA-J) used separately or in combination(data not shown).

As shown in FIG. 56A, “untreated” negative control cells, which were notexposed to CRISPR/Cpf1 or shuttle peptide, exhibited no significantgenome editing (lack of HLA-negative cells). FIGS. 56B-56D show thateach guide crRNA (RNA-E, RNA-G, RNA-J) used separately providedcomparable HLA KO efficiencies, while FIG. 56E shows the combination thethree guides crRNA enhanced the HLA KO efficiency by almost a factor oftwo. These observations were confirmed by performing a T7E1 cleavageassay as described in Example 13.4, followed by agarose gelelectrophoresis (data not shown).

G.9 Increased Cytotoxicity of NK Cells Genome-Edited to Inactive theNKG2A Gene

Genome editing was performed in NK-92 cells to evaluate whetherinactivation of the endogenous NKG2A gene could increase thecytotoxicity of the NK-92 cells. Briefly, one million NK-92 cells wereincubated with Cpf1-NLS (1.5 μM) gRNA complex targeting the NKG2A geneand with FSD23 (6 μM) for 90 sec. After transduction, cells wereincubated in complete medium with IL-2 (20 ng/mL) for 48 h at 37° C.NK-92 cells were then immunolabelled with a phycoerythrin (PE)-labelledanti-NKG2A antibody (Miltenyi Biotec # CD159a) following themanufacturer recommendations. NK-92 cells were then analyzed with FACSand scored as a function of their anti-NKG2A detection (PE fluorescence)level and the results are shown in FIG. 57A. As controls, unlabelledwild-type NK-92 cells (“unlabelled WT cells”) had no antibody signal,and labelled wild-type NK-92 cells (“labelled WT cells”) had fullimmunolabelling signal. For NKG2A-KO NK-92 cells, two cell populations(peaks) were observed: one with a complete knock-out of NKG2A receptorexpression on the cell surface (“Complete NKG2A KO cells”), and theother with a partial lack of expression (“Partial NKG2A KO cells”).

To study the effect of inactivation of the NKG2A gene on thecytotoxicity of the NK-92 cells, we evaluated the ability of WT andNKG2A KO NK-92 cells to kill target HeLa cells. The NKG2A receptorencoded by the NKG2A gene in NK cells normally binds HLA-E epitopesexpressed on the surface of potential target cells, which inhibits thecytotoxic activity of the NK cells (effector). To improve thiseffector:target cell binding, HeLa cells were treated with interferons(50 ng/mL) to increase their HLA-E cell surface expression. Prior tobeing exposed to effector NK-92 cells, interferon-treated HeLa cellswere exposed for 45 minutes at 37° C. to Calcein-AM (ThermoFisher #C3099), a non-fluorescent, hydrophobic compound that easily permeatesintact live cells. The hydrolysis of Calcein-AM by intracellularesterases produces Calcein, a hydrophilic, strongly fluorescent compoundthat is well-retained in the cell cytoplasm. HeLa cells withintracellular Calcein were then centrifuged and incubated in completemedium before being exposed to WT or NKG2A-KO NK cells in a 96-wellplate for 4 hrs at 37° C. Killing of the target HeLa cells by effectorNK cells results in release of the intracellular Calcein into theextracellular medium. The 96-well plate was then centrifuged for 5minutes at 1250 rpm and the Calcein signal in the supernatant wasanalyzed by spectrophotometry with excitation at 488 nm and detection at510 nm. Results are shown in FIG. 57B, which presents the percentage oflysis of the target HeLa cells (measured by Calcein release) as afunction of different ratios of effector NK cells to target HeLa cells(E:T ratio). The results indicate that the knock out of the NKG2Areceptor expression on the surface of NK-92 cells (“NK92 NKG2A-KO”)increased the cytotoxic activity of the effector cells as compared towild-type NK-92 cells (“NK92-WT”). More specifically, NKG2A-KO NK-92effector cells killed 10-15% more target HeLa cells than WT NK-92 cellsat the different effector:target ratios (E:T ratios) tested.

G.10 Different Rationally-Designed Peptide Shuttle Agents DeliverCRISPR/Cpf1 Systems for B2M and NKG2A Gene Editing in HeLa and NK-92Cells.

Cpf1-NLS recombinant protein was prepared as described in Example G.2. Amix composed of a Cpf1-NLS recombinant protein with a single guide crRNA(see below) targeting a nucleotide sequence of the B2M or the NKG2Agenes was co-incubated with 20 μM or 6 μM of the indicated peptide andincubated with HeLa or NK-92 cells, respectively, for 1 min in PBS,using the transduction protocols as generally described in Example 3.1a.Cells were then washed with PBS and harvested to proceed with the T7E1protocol assay as described in Example 13.4. The crRNA constructed andtheir targets were: B2M crRNA-G (SEQ ID NO: 168) and NKG2A crRNA (SEQ IDNO: 165).

Table G1 shows the percentage insertions and deletions (% INDELs)resulting from cleavage of the targeted genomic B2M and NKG2A DNAsequences with CRISPR/Cpf1 (1.33 μM) and crRNA (2 μM), which weretransduced with the indicated peptides used at different concentrationsin HeLa and NK-92 cells after T7E1 assay and direct quantification onagarose gel electrophoresis (n=2). The peptide FSD67, which failed torespect parameter (5) (low hydrophobic moment), failed to transduceCRISPR/Cpf1.

Cells Peptides Gene target % INDELs Design parameters HeLa FSD43 B2M 50OK FSD44 74 OK FSD45 45 OK FSD46 74 OK FSD47 55 OK FSD48 46 OK FSD49 27OK FSD50 63 OK FSD51 65 OK FSD61 65 OK FSD62 65 OK FSD63 70 OK FSD99 49OK NK-92 FSD115 B2M 15 OK FSD99 crRNA-G 32 OK FSD44 19 OK FSD61 23 OKFSD63 10 OK FSD67 0 Low hydrophobic moment (μH = 2.47) FSD68 8 Nohydrophobic core FSD69 23 OK FSD70 28 OK FSD71 15 OK FSD100 NKG2A 16 OKFSD101 17 OK OK = peptide sequence respects parameters (1)-(15).

Example H Rationally Designed Peptide Shuttle Agents Enable Transductionof Transcription Factor HOXB4

Human HOXB4 recombinant protein (Example 14.1) was constructed,expressed and purified from a bacterial expression system as describedin Example 1.4. THP-1 cells were cultured and tested in the proteintransduction assay as generally described in Example 3.1b. Briefly,THP-1 cells were plated at 30 000 cells/well one day beforetransduction. HOXB4-WT recombinant protein (300 nM or 50 nM) wasco-incubated with FSD10 or FSD18 (1 μM) and then exposed to THP-1 cellsfor 30 min in the presence of serum. The cells were subjected to realtime-PCR analysis as described in Example 14.2 to measure the mRNAlevels of a target gene as a marker for HOXB4 activity, which was thennormalized to the target gene mRNA levels detected in the negativecontrol cells (no treatment), to obtain a “Fold over control” value.Total RNA levels (ng/μL) were also measured as a marker for cellviability. Results are shown below.

TABLE H1 HOXB4-WT transduction by FSD10 and FSD18 in THP-1 cells Conc.Conc. of Fold over Total RNA of HOXB4- control in ng/μL Cargo/ peptideWT (mean ± (mean ± Cells peptide (μM) (μM) St. Dev) St. Dev) THP-1 Notreatment 0 0   1 ± 0.1 172 ± 9.21 HOXB4-WT 1.5 2.5 ± 0.2 175 ± 7.05alone FSD10 alone 1 0  1.1 ± 0.14 181 ± 10.7 FSD18 alone  1.5 ± 0.09 157± 3.9  FSD10 + 0.3 17.5 ± 0.21 159 ± 12.5 HOXB4-WT 0.05 15.3 ± 0.3  176± 4.71 FSD18 + 0.3 15.8 ± 0.19  154 ± 11.24 HOXB4-WT 0.05  16.7 ± 15.61154 ± 3.9 

These results show that the shuttle agents FSD10 and FSD18 are able todeliver the transcription factor HOXB4-WT to the nucleus of THP-1 cellsin the presence of serum, resulting in a dose-dependent increase in mRNAtranscription of the target gene.

Example I Co-Transduction with an Independent Fluorescent Protein MarkerEnables Isolation of Successfully Transduced Cells

The ability of domain-based and rationally-designed peptide shuttleagents described herein to co-transduce two different polypeptide cargossimultaneously is shown in Example 9.6 (i.e., co-transduction of thefluorescent proteins GFP-NLS and mCherry-NLS) and in Example G.6 (i.e.,co-transduction of the genome editing complexes CRISPR/Cas9 andCRISPR/Cpf1).

The results presented in Example I demonstrate the successfulco-transduction of a fluorescent protein marker (e.g., GFP-NLS) and agenome editing complex (e.g., CRISPR/Cpf1). Surprisingly, althoughco-transduction with the fluorescent protein marker was not found tosignificantly increase overall genome-editing efficiency per se, astrikingly high proportion of cells positive for genome-engineering werepositive for the fluorescent protein marker. Isolating cells positivefor the fluorescent protein marker resulted in a significant increase inthe proportion of successfully genome-edited cells. Furthermore, thecorrelation was found to be concentration specific in that cellpopulations exhibiting the highest fluorescence of the protein markeralso exhibited the highest proportion of successful genome-editing.

I.1 Enrichment of Genome-Edited T Cells by FACS FollowingCo-Transduction of CRIPSR/Cpf1-NLS and GFP-NLS

Cpf1-NLS recombinant protein was prepared as described in Example G.2and GFP-NLS recombinant protein was prepared as described in Example5.1.

Unless otherwise specified, T cells used herein were obtained fromhealthy human blood collected in heparinized tubes. T cells wereisolated using a Ficoll™ technique (Ficoll-Paque™ GE or Lymphoprep StemCell Technologies). Briefly, blood was mixed with the Ficoll™ solutionin conical tubes (50 mL) and centrifuged at 2280 rpm for 20 minutes.Mononuclear cells were harvested and transferred in another conical tube(50 mL) before washing with PBS and centrifugation at 1100 rpm for 10minutes. Cells were resuspended in 5 mL of PBS containing 20% FBS. Cellswere counted and then incubated in a culture medium composed by RPMIadvanced (cat 12633012 ThermoFisher), 10% FBS, 1% Penstrep (15140122ThermoFisher), 1% L-glutamine (25030081 ThermoFisher) IL-2 30 U/ml).Next, T cells were enriched with a Human T cell Enrichment Kit (StemCell# cat 19051) by negative selection following the manufacturerinstructions. The enriched T cells were validated using a specificanti-CD3 antibody (Biolegend # cat: 300438). At this step, collectedcells were typically around 99% T cells. T cells were activated byadding IL-2 at 30 U/mL and the anti-CD28 antibody (ThermoFisher # cat16-0289-85) in complete medium for 5 days prior to experimentation. Theactivation of T cell expansion was then double-checked with bothanti-CD25 and anti-CD137 antibodies.

T cells were transduced with a CRISPR/Cpf1 complex comprising a guideRNA (B2M crRNA-E, SEQ ID NO: 166) designed to cleave and inactivate theB2M gene as generally described in Example G.2, resulting in theinactivation of cell surface HLA in genome-edited cells. Briefly, 4million of activated T cells were used for each condition. In oneexperimental condition, cells were treated with a mix containing thepeptide FSD18 at 15 μM and the CRISPR/Cpf1-NLS complex just beforeincubation with cells for 90 seconds. In a second experimentalcondition, GFP-NLS (20 μM) was added to the mix containing FSD18 at 15μM and the CRISPR/Cpf1-NLS complex just before incubation with cells for90 seconds. Untreated cells were used as negative control. Aftertransduction, cells were washed and resuspended in culture media.Untreated cells and cells treated with the CRISPR/Cpf1-NLS complex wereincubated in complete media for 48 hours before flow cytometry and T7E1analysis. A first part of the cells treated with CRISPR/Cpf1-NLS andGFP-NLS complex were incubated for 48 hours in T cell medium before flowcytometry and T7E1 analysis. The second part of cells were centrifugatedand resuspended in PBS with 1% serum. GFP positive (+) and GFP negative(−) cells were separated using cell sorters based on the fluorescencesignal and fractions were collected and incubated for 48 hours in T cellmedium before flow cytometry and T7E1 analysis.

Results are shown in FIGS. 58A-58F, in which quadrant 1 (Q1) representscells that are HLA-positive (non-genome edited) and GFP-negative;quadrant 2 (Q2) represents cells that are HLA-positive (non-genomeedited) and GFP-positive; quadrant 3 (Q3) represents cells that areHLA-negative (successfully genome edited) and GFP-positive; and quadrant4 (Q4) represents cells that are HLA-negative (successfully genomeedited) and GFP-negative. Flow cytometry results based on cell size andgranularity using respectively the Forward Scatter (FSC) and the SideScatter (SSC) parameters showed that co-delivery of GFP-NLS andCRISPR/Cpf1 systems under the tested conditions did not significantlyaffect the viability of the transduced T cells (data not shown).

As shown in FIG. 58A, “untreated” negative control cells not exposed tothe peptide shuttle agent, GFP, nor CRISPR/Cpf1 resulted in 99% of cellsin Q1 (non-genome edited, GFP-negative). FIG. 58B shows that cellsexposed to the peptide FSD18 (15 μM) and CRISPR/Cpf1 in the absence ofGFP-NLS resulted in 10.1% of cells in Q4—i.e., being HLA-negative(successfully genome-edited) and GFP-negative. FIG. 58C shows that cellsexposed to both GFP-NLS and CRISPR/Cpf1 in the presence of the peptideFSD18, resulted in 65.7% (54.4%+11.3%) GFP-positive cells in Q2+Q3, and11.7% (0.441%+11.3%) HLA-negative cells (successfully genome-edited) inQ3+Q4. Comparing FIGS. 58B and 58C, the presence or absence of GFP wasnot found to significantly increase overall genome editing efficiency(10.1% versus 11.7% of HLA-negative cells). Surprisingly, it wasobserved that over 96% of genome-edited cells (HLA-negative) wereGFP-positive as well [FIG. 58C, Q3(Q3+Q4)]. Fluorescence-activated cellsorting (FIG. 58D) of cells based on their GFP-fluorescence into aGFP-negative fraction (FIG. 58E) and a GFP-positive fraction (FIG. 58F)resulted in an increase in the proportion of genome-edited(HLA-negative) cells to 29.7% in the GFP-positive fraction (FIG. 58F).Strikingly, the proportion of genome-edited (HLA-negative) cells in theGFP-negative fraction (FIG. 58E) was less than 0.1%.

Each cell fraction was then subjected to the T7E1 cleavage assay asdescribed in Example 13.4, and the different samples were subjected toagarose gel electrophoresis. The results are shown in FIG. 58G, whereinthin dashed arrows indicate the bands corresponding to the target gene,and thicker solid arrows indicate the bands corresponding to the CRISPRsystem-mediated cleavage products of this target gene, which indicatethe successful transduction of fully functional CRISPR/Cpf1-NLS genomeediting complexes. As can be seen in FIG. 58G, the GFP-positive cellfractions (“GFP+cells”; lane 4) had increased genome-editing efficiencyas compared to cells before sorting (“no cell sorting”; lane 3). PeptideFSD18 and the CRISPR/Cpf1-NLS complex with GFP-NLS (lane 3) and withoutGFL-NLS (lane 2) showed the same genome editing level, indicating thatthe addition of GFP-NLS does not affect the transduction process.GFP-negative cell fractions (lane 5), and negative control (“untreated”;lane 1) demonstrated no detectable genome editing.

I.2 Further Enrichment of Genome-Edited T Cells by FACS FollowingCo-Transduction of CRIPSR/Cpf1-NLS and GFP-NLS

The experiment described in Example I.1 was repeated on activated Tcells using 12 μM or 15 μM of the peptide FSD18. Since bothconcentrations of FSD18 produced similar results, only the results using15 μM of FSD18 are shown herein. Flow cytometry results based on cellsize and granularity using respectively the Forward Scatter (FSC) andthe Side Scatter (SSC) parameters showed that co-delivery of GFP-NLS andCRISPR/Cpf1 systems under the tested conditions did not significantlyaffect the viability of the transduced T cells (data not shown).

FIGS. 59A and 59B, show the results of “untreated” negative controlcells not exposed to the peptide shuttle agent, GFP, nor CRISPR/Cpf1,which were analyzed by flow cytometry for GFP fluorescence (FIG. 59A)and cell surface HLA expression (FIG. 59B). T cells were co-transducedwith both GFP-NLS and CRISPR/Cpf1 via the peptide FSD18 (15 μM) andsorted based on their fluorescence signal, and cell fractions werecollected and incubated for 48 hours. The resulting fraction of cellssorted for GFP fluorescence distribution is shown in FIG. 59C. The twogates in FIG. 59C indicate the fraction of cells that were considered tobe GFP-positive (“GFP+”; 93.2%) and the sub-fraction of cells that wereconsidered as exhibiting high GFP fluorescence (“GFP high”; 33.1%).Fluorescence-activated cell sorting analysis was performed to quantifythe level of cell surface HLA expression in cells considered to beGFP-positive (FIG. 59D) as compared to cells considered as exhibitinghigh GFP fluorescence (FIG. 59E). As can be seen in FIG. 59D, 21.8% ofGFP-positive cells were HLA-negative (successfully genome-edited),whereas this value rose to a striking 41.6% amongst cells exhibitinghigh GFP fluorescence (FIG. 59E). Thus, the proportion of successfullygenome-edited (HLA-negative) cells increased with the fluorescence levelof the fluorescent protein marker (GFP-NLS).

The above co-transduction experiment was repeated using 12 μM or 15 μMFSD18, followed by fluorescence-activated cell sorting into GFP-positiveand GFP-negative cell fractions. Each fraction was subjected to the T7E1cleavage assay as described in Example 13.4, and the different sampleswere subjected to agarose gel electrophoresis. Consistent with theresults of the flow cytometry experiments described above, the resultsfrom T7E1 cleavage assays showed that the GFP-positive cell fractionshad increased genome-editing efficiency as compared to in theGFP-negative cell fractions (data not shown).

I.3 Enrichment of Genome-Edited THP-1 Cells by FACS FollowingCo-Transduction of CRIPSR/Cpf1-NLS and GFP-NLS

The experiments performed in Examples 1.1 and 1.2 were reproduced inTHP-1 cells with similar results. Co-transduction of CRIPSR/Cpf1-NLS andGFP-NLS in the presence 2 μM FSD18, followed by fluorescence-activatedcell sorting of GFP-positive cells resulted in a significant enrichmentof genome edited cells (data not shown).

I.4 Successful Subsequent Transduction of Previously Sorted GFP-NegativeCells

This example shows that untransduced cells following a first round oftransduction with a peptide shuttle agent are not necessarily refractoryto subsequent transductions.

T cells obtained as described in Example I.1 were subjected to a firsttransduction by co-incubation of FSD18 (10 μM) and GFP-NLS (20 μM) for90 sec before washing and incubation at 37° C. The “untreated” negativecontrol cells showed no GFP signal (FIG. 60A). Cell sorting wasperformed 18 h after GFP-NLS transduction. GFP-positive and GFP-negativecells were separated using cell sorters based on the fluorescence signal(see FIG. 60B), and GFP-negative cells were harvested and isolated (seeFIG. 60C). A second GFP-NLS transduction was performed on theGFP-negative T cell population using the same protocol as the firsttransduction. GFP-NLS transduction was analyzed by flow cytometry aspreviously described 18 h later and GFP-positive cells were scored byflow cytometry. The results from this second transduction are shown inFIG. 60D, in which the GFP-NLS transduction efficiency was found to be60.6%.

These results indicate that untransduced cells following a first roundof transduction with a peptide shuttle agent are not necessarilyrefractory to subsequent transductions, and that overall transductionefficiency in a starting cell population may be increased by repeatedsuccessive transduction experiments on the untransduced cell fraction.This method of repeated successive transduction experiments, along withthe co-transduction results presented in Examples I.1, I.2 and I.3,suggest an attractive method for increasing genome editing efficiency invaluable cell populations (e.g., patient-derived cells for celltherapy), and/or in cell populations that are inherently more difficultto transduce.

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1.-60. (canceled)
 61. A protein transduction composition comprising ashuttle agent and a polypeptide cargo to be delivered intracellularly,wherein the shuttle agent is: (1) a peptide having an overall length ofbetween 20 and 150 amino acids comprising (2) an amphipathicalpha-helical motif having a positively-charged hydrophilic outer face,and (3) a hydrophobic outer face, wherein at least seven of thefollowing parameters (4) to (15) are respected: (4) the hydrophobicouter face comprises a highly hydrophobic core consisting of spatiallyadjacent L, I, F, V, W, and/or M amino acids representing 12 to 50% ofthe amino acids of the peptide, based on an open cylindricalrepresentation of the alpha-helix having 3.6 residues per turn; (5) thepeptide has a hydrophobic moment (μ) of 3.5 to 11; (6) the peptide has apredicted net charge of at least +4 at physiological pH; (7) the peptidehas an isoelectric point (pI) of 8 to 13; (8) the peptide is composed of35% to 65% of any combination of the amino acids: A, C, G, I, L, M, F,P, W, Y, and V; (9) the peptide is composed of 0% to 30% of anycombination of the amino acids: N, Q, S, and T; (10) the peptide iscomposed of 35% to 85% of any combination of the amino acids: A, L, K,or R; (11) the peptide is composed of 15% to 45% of any combination ofthe amino acids: A and L, provided there being at least 5% of L in thepeptide; (12) the peptide is composed of 20% to 45% of any combinationof the amino acids: K and R; (13) the peptide is composed of 0% to 10%of any combination of the amino acids: D and E; (14) the differencebetween the percentage of A and L residues in the peptide (% A+L), andthe percentage of K and R residues in the peptide (% K+R), is less thanor equal to 10%; and (15) the peptide is composed of 10% to 45% of anycombination of the amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M,N, T and H, wherein the concentration of the shuttle agent in saidcomposition is sufficient to increase transduction efficiency andcytosolic delivery of the polypeptide cargo, as compared to in theabsence of the shuttle agent, upon contact of the composition withtarget eukaryotic cells, and wherein the shuttle agent and thepolypeptide cargo comprise independent polypeptide backbones or are notcovalently bound.
 62. The protein transduction composition of claim 61,wherein: (i) said shuttle agent is a peptide having a minimum length of21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids, and a maximumlength of 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,55, 60, 65, 70, 80, 90, 100, 110, 120, 130, or 140 amino acids; (ii)said amphipathic alpha-helical motif has a hydrophobic moment (μ)between a lower limit of 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3,4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7,5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, or 7.0, andan upper limit of 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4,10.5, 10.6, 10.7, 10.8, 10.9, or 11.0; (iii) said amphipathicalpha-helical motif comprises a positively-charged hydrophilic outerface comprising: (a) at least two, three, or four adjacentpositively-charged K and/or R residues upon helical wheel projection;and/or (b) a segment of six adjacent residues comprising three to five Kand/or R residues upon helical wheel projection, based on an alpha helixhaving angle of rotation between consecutive amino acids of 100 degreesand/or an alpha-helix having 3.6 residues per turn; (iv) saidamphipathic alpha-helical motif comprises a hydrophobic outer facecomprising: (a) at least two adjacent L residues upon helical wheelprojection; and/or (b) a segment often adjacent residues comprising atleast five hydrophobic residues selected from: L, I, F, V, W, and M,upon helical wheel projection, based on an alpha helix having angle ofrotation between consecutive amino acids of 100 degrees and/or analpha-helix having 3.6 residues per turn; (v) said hydrophobic outerface comprises a highly hydrophobic core consisting of spatiallyadjacent L, I, F, V, W, and/or M amino acids representing from 12.5%,13%, 13.5%, 14%, 14.5%, 15%, 15.5%, 16%, 16.5%, 17%, 17.5%, 18%, 18.5%,19%, 19.5%, or 20%, to 25%, 30%, 35%, 40%, or 45% of the amino acids ofthe peptide; (vi) said peptide has a hydrophobic moment (μ) between alower limit of 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0,5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4,6.5, 6.6, 6.7, 6.8, 6.9, or 7.0, and an upper limit of 9.5, 9.6, 9.7,9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, or 10.5; (vii) said peptide hasa predicted net charge of from +4, +5, +6, +7, +8, or +9, to +10, +11,+12, +13, +14, or +15; (viii) said peptide has a predicted pI of 10-13;or (ix) any combination of (i) to (viii).
 63. The protein transductioncomposition of claim 61, wherein said shuttle agent respects at leastone, at least two, at least three, at least four, at least five, atleast six, or all of the following parameters: (1) the peptide iscomposed of from 36% to 64%, 37% to 63%, 38% to 62%, 39% to 61%, or 40%to 60% of any combination of the amino acids: A, C, G, I, L, M, F, P, W,Y, and V; (2) the peptide is composed of from 1% to 29%, 2% to 28%, 3%to 27%, 4% to 26%, 5% to 25%, 6% to 24%, 7% to 23%, 8% to 22%, 9% to21%, or 10% to 20% of any combination of the amino acids: N, Q, S, andT; (3) the peptide is composed of from 36% to 80%, 37% to 75%, 38% to70%, 39% to 65%, or 40% to 60% of any combination of the amino acids: A,L, K, or R; (4) the peptide is composed of 15% to 40%, 20% to 40%, 20 to35%, or 20 to 30% of any combination of the amino acids: A and L; (5)the peptide is composed of 20% to 40%, 20 to 35%, or 20 to 30% of anycombination of the amino acids: K and R; (6) the peptide is composed of5 to 10% of any combination of the amino acids: D and E; (7) thedifference between the percentage of A and L residues in the peptide (%A+L), and the percentage of K and R residues in the peptide (% K+R), isless than or equal to 9%, 8%, 7%, 6%, or 5%; and (8) the peptide iscomposed of 15% to 40%, 20% to 35%, or 20% to 30% of any combination ofthe amino acids: Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T, and H. 64.The protein transduction composition of claim 61, wherein said shuttleagent further comprises a histidine-rich domain positioned towards the Nterminus and/or towards the C terminus of the shuttle agent, whereinsaid histidine-rich domain is a stretch of at least 3, at least 4, atleast 5, or at least 6 amino acids comprising at least 50%, at least55%, at least 60%, at least 65%, at least 70%, at least 75%, at least80%, at least 85%, or at least 90% histidine residues; and/or comprisesat least 2, at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, or at least 9 consecutive histidine residues.
 65. Theprotein transduction composition of claim 61, wherein said shuttle agentfurther comprises a flexible linker domain rich in serine and/or glycineresidues.
 66. The protein transduction composition of claim 61, whereinsaid shuttle agent comprises or consists of the amino acid sequence of:(a) [X1]-[X2]-[linker]-[X3]-[X4]  (Formula 1);(b) [X1]-[X2]-[linker]-[X4]-[X3]  (Formula 2);(c) [X2]-[X1]-[linker]-[X3]-[X4]  (Formula 3);(d) [X2]-[X1]-[linker]-[X4]-[X3]  (Formula 4);(e) [X3]-[X4]-[linker]-[X1]-[X2]  (Formula 5);(f) [X3]-[X4]-[linker]-[X2]-[X1]  (Formula 6);(g) [X4]-[X3]-[linker]-[X1]-[X2]  (Formula 7); or(h) [X4]-[X3]-[linker]-[X2]-[X1]  (Formula 8), wherein: [X1] is selectedfrom: 2[Φ]−1[+]−2[Φ]−1[ζ]−1[+]−; 2[Φ]−1[+]−2[Φ]−2[+]−;1[+]−1[Φ]−1[+]−2[Φ]−1[ζ]−1[+]−; and 1[+]−1[Φ]−1[+]−2[Φ]−2[+]−; [X2] isselected from: −2[Φ]−1[+]−2[Φ]−2−[ζ]−; −2[Φ]−1[+]−2[Φ]−2[+]−;−2[Φ]−1[+]−2[Φ]−1[+]−1[ζ]−; −2[Φ]−1[+]−2[Φ]−1[ζ]−1[+]−;−2[Φ]−2[+]−1[Φ]−2[+]−; −2[Φ]−2[+]−1[Φ]−2[ζ]−;−2[Φ]−2[+]−1[Φ]−1[+]−1[ζ]−; and −2[Φ]−2[+]−1[Φ]−1[ζ]−1[+]−; [X3] isselected from: −4[+]−A−; −3[+]−G−A−; −3[+]−A−A−; −2[+]−1[Φ]−1[+]−A−;−2[+]−1[Φ]−G−A−; −2[+]−1[Φ]−A−A−; or −2[+]−A−1[+]−A; −2[+]−A−G−A;−2[+]−A−A−A−; −1[Φ]−3[+]−A−; −1[Φ]−2[+]−G−A−; −1[Φ]−2[+]−A−A−;−1[Φ]−1[+]−1[Φ]−1[+]−A; −1[Φ]−1[+]−1[Φ]−G−A; −1[Φ]−1[+]−1[Φ]−A−A−;1[Φ]−1[+]−A−1[+]−A; −1[Φ]−1[+]−A−G−A; −1[Φ]−1[+]−A−A−A;−A−1[+]−A−1[+]−A; −A−1[+]−A−G−A; and −A−1[+]−A−A−A; [X4] is selectedfrom: −1[ζ]−2A−1[+]−A; −1[ζ]−2A−2[+]; −1[+]−2A−1[+]−A;−1[ζ]−2A−1[+]−1[ζ]−A−1[+]; −1[ζ]−A−1[ζ]−A−1[+]; −2[+]−A−2[+];−2[+]−A−1[+]−A; −2[+]−A−1[+]−−1[ζ]−A−1[+]; −2[+]−1[ζ]−A−1[+];−1[+]−1[ζ]−A−1[+]−A; −1[+]−1[ζ]−A−2[+]; −1[+]−1[ζ]−A−1[+]−1[ζ]−A−1[+];−1[+]−2[ζ]−A−1[+]; −1[+]−2[ζ]−2[+]; −1[+]−2[ζ]−1[+]−A;−1[+]−2[ζ]−1[+]−1[ζ]−A−1[+]; −1[+]−2[ζ]−1[ζ]−A−1[+]; −3[ζ]−2[+];−3[ζ]−1[+]−A; −3[ζ]−1[+]−1[ζ]−A−1[+]; −1[ζ]−2A−1[+]−A; −1[ζ]−2A−2[+];−1[ζ]−2A−1[+]−1[ζ]−A−1[+]; −2[+]−A−1[+]−A; −2[+]−1[ζ]−1[+]−A;−1[+]−1[ζ]−A−1[+]−A; −1[+]−2A−1[+]−1[ζ]−A−1[+]; and −1[ζ]−A−1[ζ]−A−1[+];and [linker] is selected from: -Gn-; −Sn-; −(GnSn)n-; −(GnSn)nGn-;−(GnSn)nSn-; −(GnSn)nGn(GnSn)n-; and (GnSn)nSn(GnSn)n-; wherein: [Φ] isan amino acid which is: Leu, Phe, Trp, Ile, Met, Tyr, or Val; [+] is anamino acid which is: Lys or Arg; [ζ] is an amino acid which is: Gln,Asn, Thr, or Ser; A is the amino acid Ala; G is the amino acid Gly; S isthe amino acid Ser; and n is an integer from 1 to 20, 1 to 19, 1 to 18,1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1 to 12, 1 to 11, 1 to 10,1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4, or 1 to
 3. 67. Theprotein transduction composition of claim 61, wherein said polypeptidecargo lacks a cell penetrating domain.
 68. The protein transductioncomposition of claim 61, wherein said polypeptide cargo comprises a cellpenetrating domain.
 69. The protein transduction composition of claim61, wherein said polypeptide cargo comprises a subcellular targetingdomain which is: (a) a nuclear localization signal (NLS); (b) anucleolar signal sequence; (c) a mitochondrial signal sequence; or (d) aperoxisome signal sequence.
 70. The protein transduction composition ofclaim 61, wherein said polypeptide cargo is complexed with a DNA and/orRNA molecule.
 71. The protein transduction composition of claim 61,wherein said polypeptide cargo is a transcription factor, a nuclease, acytokine, a hormone, a growth factor, an antibody, a peptide cargo, anenzyme, an enzyme inhibitor, or any combination thereof.
 72. The proteintransduction composition of claim 71, wherein: (a) said transcriptionfactor is: HOXB4, NUP98-HOXA9, Oct3/4, Sox2, Sox9, Klf4, c-Myc, MyoD,Pdx1, Ngn3, MafA, Blimp-1, Eomes, T-bet, FOXO3A, NF-YA, SALL4, ISL1,FoxA1, Nanog, Esrrb, Lin28, HIF1-alpha, Hlf, Runx1t1, Pbx1, Lmo2, Zfp37,Prdm5, Bcl-6, or any combination thereof; (b) said nuclease is acatalytically active or catalytically dead: RNA-guided endonuclease,CRISPR endonuclease, type I CRISPR endonuclease, type II CRISPRendonuclease, type III CRISPR endonuclease, type IV CRISPR endonuclease,type V CRISPR endonuclease, type VI CRISPR endonuclease, CRISPRassociated protein 9 (Cas9), Cpf1, CasY, CasX, zinc-finger nuclease(ZFNs), Transcription activator-like effector nucleases (TALENs), homingendonuclease, meganuclease, DNA-guided nuclease, Natronobacteriumgregoryi Argonaute (NgAgo), or any combination thereof; (c) saidantibody recognizes an intracellular antigen; and/or (d) said peptidecargo recognizes an intracellular molecule.
 73. A protein transductionagent comprising the shuttle agent as defined in claim
 61. 74. A proteintransduction agent comprising the shuttle agent as defined in claim 62.75. A protein transduction agent comprising the shuttle agent as definedin claim
 63. 76. A protein transduction agent comprising the shuttleagent as defined in claim
 64. 77. A protein transduction agentcomprising the shuttle agent as defined in claim
 65. 78. A proteintransduction agent comprising the shuttle agent as defined in claim 66.79. A kit for protein transduction, the kit comprising a shuttle agentand a polypeptide cargo to be delivered intracellularly, wherein theshuttle agent is as defined in claim
 61. 80. A kit for proteintransduction, the kit comprising a shuttle agent and a polypeptide cargoto be delivered intracellularly, wherein the shuttle agent is as definedin claim
 62. 81. A kit for protein transduction, the kit comprising ashuttle agent and a polypeptide cargo to be delivered intracellularly,wherein the shuttle agent is as defined in claim
 63. 82. A kit forprotein transduction, the kit comprising a shuttle agent and apolypeptide cargo to be delivered intracellularly, wherein the shuttleagent is as defined in claim
 64. 83. A kit for protein transduction, thekit comprising a shuttle agent and a polypeptide cargo to be deliveredintracellularly, wherein the shuttle agent is as defined in claim 65.84. A kit for protein transduction, the kit comprising a shuttle agentand a polypeptide cargo to be delivered intracellularly, wherein theshuttle agent is as defined in claim 66.